Superagonists, partial agonists and antagonists of interleukin-2

ABSTRACT

Novel human interleukin-2 (IL-2) muteins or variants thereof are provided. In particular, provided are IL-2 muteins that have an increased binding capacity for IL-2Rβ receptor and a decreased binding capacity for IL-2Rγc receptor, as compared to wild-type IL-2. Such IL-2 muteins are useful, for example, as IL-2 partial agonist and antagonists in applications where reduction or inhibition of one or more IL-2 and/or IL-15 functions is useful (e.g., in the treatment of graft versus host disease (GVHD) and adult T cell leukemia). Also provided are nucleic acids encoding such IL-2 muteins, methods of making such IL-2 muteins, pharmaceutical compositions that include such IL-2 muteins and methods of treatment using such pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/930,057, filed May 12, 2020, which is a continuation of U.S.application Ser. No. 16/175,709, filed Oct. 30, 2018, which is adivisional of U.S. application Ser. No. 15/305,831, filed Oct. 21, 2016,allowed, which is a National Stage Entry of International PatentApplication No. PCT/US2015/027635, filed Apr. 24, 2015, which claims thebenefit of priority to U.S. Provisional Application No. 61/983,973,filed Apr. 24, 2014, the contents of each are incorporated herein byreference in their entireties.

This invention was made with U.S. Government support under Grant Nos.AI051321 and DK094541 awarded by the National Institutes of Health. TheU.S. Government has certain rights in this invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM,LISTING APPENDIX SUBMITTED ON A COMPACT DISK

This invention incorporated by reference the Sequence Listing text copysubmitted herewith, which was created on Jun. 21, 2017, entitled068597-5029-US02_Sequence_Listing.txt which is 48 kilobytes in size.

BACKGROUND

Interleukin 2 (IL-2) is a pluripotent cytokine produced primarily byactivated CD4⁺ T cells, which plays a crucial role in producing a normalimmune response. IL-2 promotes proliferation and expansion of activatedT lymphocytes, potentiates B cell growth, and activates monocytes andnatural killer cells. It was by virtue of these activities that IL-2 wastested and is used as an approved treatment of cancer (aldesleukin,Proleukin®).

In eukaryotic cells, human IL-2 is synthesized as a precursorpolypeptide of 153 amino acids, from which 20 amino acids are removed togenerate mature secreted IL-2 (Taniguchi 1983). Recombinant human IL-2has been produced in E. coli (Rosenberg 1984), in insect cells (Smith1985) and in mammalian COS cells (Taniguchi 1983).

Interleukin-2 (IL-2) is a four α-helical bundle type I cytokine firstidentified as a T cell growth factor (Morgan et al., Science 193: 1007(1976)) but subsequently shown to have broad actions. IL-2 promotes Thelper differentiation (Zhu et al., Annual review of immunology 28: 445(2010); Liao et al., Nat Immunol 9: 1288 (2008); and Liao et al., NatImmunol 12: 551 (2011)) and the development of regulatory T (Treg) cells(Cheng et al., Immunol Rev 241: 63 (2011)), induces natural killer andlymphokine activated killer activity (Liao et al., Immunity 38: 13(2013)), and mediates activation-induced cell death (AICD) (Lenardo etal., Nature 353: 858 (1991)).

IL-2 works by interacting with three different receptors: theinterleukin 2 receptor alpha (IL-2Rα; CD25), the interleukin 2 receptorbeta (IL-2Rβ; CD122), and the interleukin 2 receptor gamma (IL-2Rγ;CD132; common gamma chain). The first receptor to be identified was theIL-2Rα, which is a 55 kD polypeptide (p55) that appears upon T cellactivation and was originally called Tac (for T activation) antigen. TheIL-2Rα binds IL-2 with a K_(d) of approximately 10⁻⁸ M, and is alsoknown as the “low affinity” IL-2 receptor. Binding of IL-2 to cellsexpressing only the IL-2Rα does not lead to any detectable biologicresponse.

IL-2 signals via intermediate affinity receptors (K_(d)˜10⁻⁹ M) onresting T cells and NK cells that consist of IL-2Rβ and the commoncytokine receptor γ chain, γ_(c) (IL-2Rγ), or via high affinityreceptors (K_(d)˜10⁻¹¹ M) on activated lymphocytes and Treg cells, whichadditionally express IL-2Rα (CD25)(Lenardo et al., Nature 353: 858(1991); and Yuan et al., Immunol Rev 259: 103 (2014)). Whereas γ_(c) isshared by the receptors for IL-4, IL-7, IL-9, IL-15, and IL-21 (Leonardet al., Nature Reviews Immunology 1: 200 (2001)) and encoded by the genemutated in humans with X-linked severe combined immunodeficiency(Noguchi et al., Cell 73: 147 (Apr. 9, 1993)), IL-2Rβ is shared by thereceptor for IL-15 (Waldmann, Nature Reviews Immunology 6: 595 (2006)),a cytokine that is critical for normal development of NK cells andmemory CD8⁺ T cells (Waldmann, Nature Reviews Immunology 6: 595 (2006)).

The three dimensional structures of IL-2 and IL-15 bound to theirreceptors provide insights into receptor assembly and signaling (Wang etal., Science 310: 1159 (2005); and Ring et al., Nat Immunol 13: 1187(2012)). In addition to their physiological roles in the normal immuneresponse, IL-2 and IL-15 can promote pathologic responses, and atherapeutic goal is to maintain desired actions of these cytokines whileblocking untoward autoimmune or immunosuppressive responses. Twomonoclonal antibodies (mAbs) to human IL-2Rα, Daclizumab andBasiliximab, are approved by the FDA and exhibit efficacy in renaltransplantation rejection (Vincenti et al., N Engl J Med 338: 161(1998)), cardiac transplantation (Hershberger et al., N Engl J Med 352:2705 (2005)), multiple sclerosis (Gold et al., Lancet 381: 2167 (2013)),and asthma (Bielekova et al., Proc Natl Acad Sci USA 101: 8705 (2004);and Busse et al., Am J Respir Crit Care Med 178: 1002 (2008)) but theydo not block IL-2 signaling via intermediate affinity IL-2Rβ-γ_(c)receptors expressed on NK and memory CD8⁺ cells and cannot block IL-15signaling (Tkaczuk et al., Am J Transplant 2: 31 (2002)). Althoughanti-human IL-2Rβ mAb Mikβ1 can block trans-presentation of IL-2 andIL-15 to cells expressing IL-2Rβ-γ_(c) receptors (Morris et al., ProcNatl Acad Sci USA 103: 401 (2006)), it is relatively ineffective inblocking cis-signaling by IL-2 or IL-15 via their high affinityheterotrimeric receptor complexes (Morris et al., Proc Natl Acad Sci USA103: 401 (2006); and Waldmann et al., Blood 121: 476 (2013)). As such,new IL-2 muteins that can block one or more IL-2 and/or IL-15 functionsare needed. The present disclosure provides novel IL-2 muteins thatfunction as IL-2 partial agonists and antagonists.

SUMMARY

IL-2 exerts a wide spectrum of effects on the immune system, and itplays crucial roles in regulating both immune activation andhomeostasis. As an immune system stimulator, IL-2 has found use in thetreatment of cancer and chronic viral infections. The stimulatoryaffects of IL-2 can also cause havoc, mediating autoimmunity andtransplant rejection. Because of its instrumental role in immuneregulation and disease, the identification of new IL-2 molecules such asIL-2 partial agonists and antagonists remains an active area ofresearch.

Provided herein are novel IL-2 compositions based on new insights intohow IL-2 interacts with its cognate receptors. In most circumstances,IL-2 works through three different receptors: the IL-2Rα, the IL-2Rβ,and the IL-2Rγ. Most cells, such as resting T cells, are not responsiveto IL-2 since they only express the IL-2Rβ, and the IL-2Rγ, which havelow affinity for IL-2. Upon stimulation, resting T cells express therelatively high affinity IL-2 receptor IL-2Rα. Binding of IL-2 to theIL-2Rα causes this receptor to sequentially engage the IL-2Rβ, and theIL-2Rγ, bringing about T cell activation.

IL-2 “superkines” with augmented action due to enhanced binding affinityfor IL-2Rβ were previously developed (Levin et al., Nature 484: 529(2012)). It was hypothesized that this high-affinity superkine/IL-2Rβcomplex could serve as a dominant-negative scaffold to create a“receptor signaling clamp” to block endogenous signaling. Directedmutation of these super-IL-2 “full agonists” to diminish binding toIL-2Rγ_(c) would attenuate IL-2Rβ-γ_(c) heterodimerization and representa new class of mechanism-based IL-2 partial agonists and non-signaling(neutral) molecules that functionally act as antagonists by blockingendogenous cytokines and exerting no action of their own (see schematicin FIG. 1 ).

Novel human interleukin-2 (IL-2) muteins or variants thereof areprovided herein. In particular, provided are IL-2 muteins that have anincreased binding capacity for IL-2Rβ receptor and a decreased bindingcapacity to IL-2Rγ_(c) receptor. Such IL-2 muteins find use, forexample, as IL-2 partial agonists and antagonists in applications wherereduction or inhibition of one or more IL-2 and/or IL-15 functions isuseful (e.g., in the treatment of graft versus host disease (GVHD) andadult T cell leukemia). Also provided are nucleic acids encoding suchIL-2 muteins, methods of making such IL-2 muteins, pharmaceuticalcompositions that include such IL-2 muteins and methods of treatmentusing such IL-2 muteins.

In one aspect, provided herein is an IL-2 mutein having a greaterbinding affinity for IL-2Rβ and a reduced binding affinity forIL-2Rγ_(c) receptor as compared to wild-type human IL-2 (hIL-2). In someembodiments, the IL-2 mutein comprises: (a) one or more amino acidsubstitutions that increase IL-2Rβ binding affinity, selected from aminoacid positions 24, 65, 74, 80, 81, 85, 86, 89, 92, and/or 93, numberedin accordance with wild-type hIL-2; and (b) one or more amino acidsubstitutions that decrease IL-2Rγ_(c) receptor binding affinityselected from amino acid positions 18, 22, 126, and/or 130, numbered inaccordance with wild-type hIL-2.

In various embodiments, the amino acid substitutions that increaseIL-2Rβ binding affinity comprise: Q74N, Q74H, Q74S, L80F, L80V, R81D,R81T, L85V, I86V, I89V, and/or 193 or combinations thereof. In certainembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity comprise: L80F, R81D, L85V, I86V and I92F. In some embodiments,the amino acid substitutions that increase IL-2Rβ binding affinitycomprise: N74Q, L80F, R81D, L85V, I86V, I89V, and I92F. In someembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity comprise: Q74N, L80V, R81T, L85V, I86V, and I92F. In certainembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity comprise: Q74H, L80F, R81D, L85V, I86V and I92F. In someembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity comprise: Q74S, L80F, R81D, L85V, I86V and I92F. In certainembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity comprise: Q74N, L80F, R81D, L85V, I86V and I92F. In certainembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity comprise: Q74S, R81T, L85V, and 192F.

In some embodiments, the amino acid substitutions that decrease IL-2Rγ_(c) receptor binding affinity comprises amino acid substitutions L18R,Q22E, A126T and/or S130R or combinations thereof. In specificembodiments, the amino acid substitutions that decrease IL-2Rγ_(c)receptor binding affinity comprises Q126T. In certain embodiments, theamino acid substitutions that decrease IL-2Rγ_(c) receptor bindingaffinity comprises L18R and Q22E. In some embodiments, the amino acidsubstitutions that decrease IL-2R γ_(c) receptor binding affinitycomprises L18R, Q22E, and Q126T. In certain embodiments, the amino acidsubstitutions that decrease IL-2Rγ_(c) receptor binding affinitycomprises L18R, Q22E, Q126T and S130R.

In one embodiment, the IL-2 mutein having a greater binding affinity forIL-2Rβ and a reduced binding affinity for IL-2R γ_(c) receptor ascompared to wild-type human IL-2, wherein the IL-2 mutein comprises theamino acid substitutions L80F, R81D, L85V, I86V, I92F, and Q126T.

In one embodiment, the IL-2 mutein having a greater binding affinity forIL-2Rβ and a reduced binding affinity for IL-2R γ_(c) receptor ascompared to wild-type human IL-2, wherein the IL-2 mutein comprises theamino acid substitutions L18R, Q22E, L80F, R81D, L85V, 186V and I92F.

In one embodiment, the IL-2 mutein having a greater binding affinity forIL-2Rβ and a reduced binding affinity for IL-2Rγ_(c) receptor ascompared to wild-type human IL-2, wherein the IL-2 mutein comprises theamino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, Q126T andI92F.

In one embodiment, the IL-2 mutein having a greater binding affinity forIL-2Rβ and a reduced binding affinity for IL-2Rγ_(c) receptor ascompared to wild-type human IL-2, wherein the IL-2 mutein comprises theamino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F,Q126T, and S130R.

In certain embodiments, the subject IL-2 mutein has a reduced capabilityto stimulate STAT5 phosphorylation in an IL-2Rβ+ T cell as compared towild-type hIL-2. In some embodiments, the T cell is a CD8+ T cell.

In some embodiments, the subject IL-2 mutein has a reduced capability tostimulate the pERK1/ERK2 signaling in a IL-2Rβ+ cell as compared towild-type hIL-2.

In certain embodiments, the subject IL-2 mutein is an IL-2 and/or IL-15antagonist. In some embodiments, the IL-2 mutein is an inhibitor of IL-2and/or IL-15 STAT5 phosphorylation in CD8+ T cells. In some embodiments,the IL-2 mutein is an inhibitor of IL-2 and/or IL-15 inducedproliferation of CD8+ T cells. In some embodiments, the IL-2 mutein isan inhibitor of IL-2 dependent, TCR-induced cell proliferation. In oneembodiment, the IL-2 mutein is an inhibitor of IL-2 dependent Th1, Th9and/or Treg differentiation. In certain embodiments, the IL-2 mutein isa promoter of Th17 differentiation. In some embodiments, the mutein isan inhibitor of IL-2 dependent activation of NK cells.

In another aspect, provided herein is an IL-2 mutein fusion proteincomprising any one of the IL-muteins described herein linked to a humanFc antibody fragment.

In another aspect, provided herein is a pharmaceutical compositioncomprising any one of the IL-2 muteins or the IL-2 mutein fusion proteindescribed herein and a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition comprises an IL-2 muteinhaving the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V,I92F, Q126T, and S130R.

In yet another aspect, provided herein is a method of treating a subjecthaving graft versus host disease (GVHD). In various embodiments, themethod comprises administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising any one ofthe IL-2 muteins disclosed herein. In some embodiments, thepharmaceutical composition comprises an IL-2 mutein having the aminoacid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, andS130R.

In another aspect, provided herein is a method of treating a subjecthaving adult T-cell leukemia. In certain embodiments, the methodcomprises administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising any one of the IL-2muteins disclosed herein. In some embodiments, the pharmaceuticalcomposition comprises an IL-2 mutein having the amino acid substitutionsL18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic for generating subject IL-2 muteins providedherein. Left, On IL-2, the green region indicates the previouslyreported changes to generate H9 “super-IL-2” with high-affinity bindingto IL-2Rβ, thereby blocking the binding of endogenous IL-2. The redcircle indicates changes in IL-2 that decrease binding to IL-2R γ_(c)and IL-2Rβ-γ_(c) heterodimerization. Right, Depending on the level ofdisruption of IL-2R γ_(c) binding, different levels of activity andfunction should be generated, as indicated.

FIG. 2A-2B shows a FACS profile of an IL-2 mutein library as describedherein. Products of error prone PCR of the human IL-2 gene weresubjected to selection. The first generation IL-2 library was generatedthrough six rounds of selection. The first round was performed usingtetrameric IL-2Rβ coupled to phycoerythrin (PE) to bind yeast expressingIL-2 muteins (A). Subsequent rounds of selection were accomplished usingmonomeric IL-2 Rβ labeled with PE. (B) Results from the secondgeneration IL-2 library.

FIG. 3 depicts the amino acid residues altered in the IL-2 muteins withhigh-affinity IL-2Rβ binding, shown relative to the wild-type IL-2sequence. The binding affinity of each mutein and IL-2 for the IL-2Rβ isalso shown.

FIG. 4A-4B shows the stimulatory effects of IL-2 muteins withhigh-affinity binding to IL-2Rβ on CD25⁻ and CD25⁺ natural killer (NK)cells. Dose response relationships of wild-type IL-2 and the IL-2muteins 6-6, D10, and H9 on STAT5 phosphorylation witnessed in treated(A) CD25⁻ and (B) CD25⁺ YT-1 NK cells. Circles wild-type IL-2; squares6-6; triangles up H9; triangles down D10.

FIG. 5A-5B shows the CD25 independence of IL-2 mutein binding. Doseresponse curves of STAT5 phosphorylation for CD25⁻ and CD25⁺ YT-1 NKcells. (A) IL-2 and IL-2 (F42A) (circles, solid line wild-type IL-2,CD25+ cells; squares, solid line IL-2 F42A, CD25+ cells; triangles up,dashed lines wild-type IL-2, CD25− cells; triangles down, dashed line,IL-2 F42A, CD25− cells). (B) H9 and H9(F42A) (circles, solid linewild-type H9, CD25+ cells; squares, solid line H9 F42A, CD25+ cells;triangles up, dashed lines H9, CD25− cells; triangles down, dashed line,H9 F42A, CD25− cells). While the F42A mutation right shifted thedose-response curve of wild-type IL-2 on CD25⁺ cells, but had noobservable effect on CD25, the dose response curves for H9 and H9 F42Awere essentially overlapping, regardless of CD25 expression.

FIG. 6 depicts the ability of several IL-2 muteins “super agonists”(i.e., muteins with high-affinity binding to IL-2Rβ) to stimulate Tcells in the absence of the IL-2Rα. T cells isolated from CD25 knockoutmice were stimulated with an IL-2 mutein or wild-type IL-2. Doseresponse curves and respective EC50 of IL-2 muteins are provided. Asshown, all of the tested IL-2 muteins resulted in relatively increased Tcell stimulation, in the absence of the IL-2Rα, relative to wild-typeIL-2.

FIG. 7 is a FACS analysis comparing the relative ability of IL-2 muteins“super agonists” to induce experienced T cell stimulation. T cells werestimulated with two concentrations (10 ng/ml or 1 ng/ml) of IL-2 muteinor wild-type IL-2. The percentage of stimulated T cells is shown in eachFACS profile.

FIG. 8 shows the effect of IL-2 “super agonist” mutein D10 on naturalkiller (NK) cell function, specifically spontaneous andantibody-dependent cell mediated cytotoxicity. Natural killer cells(effectors) and Cr⁵¹ labeled tumor cells (targets) were incubatedtogether for 5 hours in the presence of wild-type IL-2 or the IL-2mutein D10, with or without the anti-EGFR antibody cetuximab. D10stimulation of NK cell spontaneous cytotoxicity was superior to highdose IL-2 (*p=0.008, **p=0.001) with minimal spontaneous cytotoxicitywithout IL-2 or D10 stimulation. Further, addition of D10 enhanced theADCC of the cetuximab antibody.

FIG. 9 shows the crystal structure of D10. An initial hydrophobic coremutation of L85V led to a second generation IL-2 library targetingmultiple hydrophobic core residues and a high affinity consensussequence. The crystal structure of D10 contained well-defined electrondensity in the loop region preceding helix C.

FIG. 10A-10B shows that IL-2 “super agonist” muteins with high-affinitybinding to IL-2Rβ exhibit enhanced stimulation of CD8⁺ T cells but notTregs relative to IL-2. (A) Total cell counts of host CD3⁺ CD8⁺CD44^(high) memory-phenotype (MP) T cells and (B) host CD3⁺ CD4⁺CD25^(high) T cells (regulatory T cells) was determined in the spleensof mice receiving either PBS, 20 μg IL-2, 20 μg H9, or 1.5 μgIL-2/anti-IL-2 monoclonal antibody complexes (IL-2/mAb).

FIG. 11A-11B shows that IL-2 mutein agonists with high-affinity bindingto IL-2Rβ exhibit enhanced anti-tumor response with reduced adverseeffects relative to IL-2. Pulmonary edema (pulmonary wet weight) servedas a measure of adverse toxic effects following IL-2 treatment, and wasdetermined by weighing the lungs before and after drying (A). P valuesrefer to comparisons between treatment modalities. *, p<0.05; **,p<0.01. (B) Anti-tumor properties of IL-2 muteins were tested in vivousing B16F10 melanoma cells. C57Bl/6 mice (n=3-4 mice/group) wereinjected subcutaneously with 106 B16F10 melanoma cells followed by dailyinjections of either PBS, 20 μg IL-2, 20 μg H9, or 1.5 μg IL-2/anti-IL-2monoclonal antibody complexes (IL-2/mAb) for five days once tumornodules became visible and palpable, which typically corresponded to day4 to 5 after tumor cell injections or a tumor size of about 15 mm².Shown is mean tumor area in mm² (+/−SD) vs. time upon tumor inoculation.P values refer to comparison of IL-2 with the other treatmentmodalities.

FIG. 12A-12C shows the generation of mechanism-based IL-2 muteins bydisrupting γ_(c) binding. (A) Structure of the H9-IL-2Rβ-γ_(c) complex(H9 is green; IL-2Rβ is blue; γ_(c) is gold). The mutations (L18R, Q22E,Q126T, and S130R) incorporated into H9-RETR to disrupt the interactionof IL-2 with γ_(c) are shown in cyan (right part of panel). (B, C)Surface plasmon resonance analysis of the binding of H9-IL-2Rβ (C) andH9-RET-IL-2Rβ (D) complexes to γ_(c).

FIG. 13A-13H shows the attenuated signaling of IL-2 muteins with reducedbinding affinity for IL2Rγ_(c). (A, B) Wild-type IL-2 or various H9variants were assayed for their ability to induce STAT5 phosphorylationin CD25 (A) and CD25⁺ (B) YT-1 human NK-like cells. (C, D)Internalization kinetics of IL-2Rβ (C) and IL-2Rγ (D) relative tomaximal surface expression in CD25⁻ YT-1 cells following cytokinestimulation. (E) Freshly isolated (upper panels) or pre-activated (lowerpanels) CD8⁺ T-cells were left unstimulated or stimulated with 1 μg/mlIL-2, H9-RE, H9-T, H9-RET, or H9-RETR for 30 min. CD25 expression (leftpanels) or pSTAT5 (right panels) were assayed by flow cytometry. (F)Cells as indicated were treated with IL-2, H9, H9-RET, or H9-RETR, lysedand western blotted with antibodies to pSTAT5 or total STAT5. (G)Dose-response curves of pSTAT5 induced by wild type IL-2, H9, H9-RE,H9-T, H9-RET, and H9-RETR. (H) Dose-response curves of phospho-S6ribosomal protein (pS6) by the wild type IL-2, H9, H9-RET, and H9-RETR.MFI, mean fluorescent intensity. For dose-response experiments (A, B, G,H), the abscissa indicates the log of cytokine concentration in ng/ml.MFI, mean fluorescent intensity. Data are representative of at least twoexperiments per panel (error bars, S.E.M. of triplicates).

FIG. 14A-14B shows the attenuated signaling of IL-2 muteins with reducedbinding affinity for IL2Rγ_(c) in two different assays. (A)Dose-response curves of phospho-ERK1/ERK2 protein on CD25⁻ YT1 humanNK-like cells. (B) Comparison of IL-2Rβ and IL-2Rγ expression on freshlyisolated (upper panels) or pre-activated (lower panels) human CD8⁺ Tcells. Data are representative of at least two experiments per panel.Error bars represent SEM. of triplicates.

FIG. 15 shows the proliferation and CD25 expression in response to IL-2,H9, H9-RE, H9-T, H9-RET, and H9-RETR. Induction of proliferation offreshly isolated human CD8⁺ T cells by IL-2 and H9 but not by H9-RET orH9-RETR, with intermediate effects of H9-T. Cells were labeled withCFSE, stimulated with the indicated IL-2 variants, and CFSE dilution wasassessed by flow cytometry 5 days later.

FIG. 16A-16F shows the effects of H9-RET and RETR on proliferation,STAT5 binding, and gene expression. (A) Freshly-isolated andpre-activated human CD8⁺ T cells were cultured in 96-well plates withvarying concentrations of indicated IL-2 variants for 2 days, and[³H]-thymidine incorporation measured. Bars represent mean f S.E.M.Cells were combined from 2 individual donors. Data are representative ofat least two independent experiments. (B) RNA-Seq heat map analysis ofpre-activated CD8⁺ T cells treated with IL-2, H9, H9-RET, and H9-RETR (1μg/ml) for 24 hr. Shown are the genes whose expression is eitherupregulated or downregulated by IL-2 relative to the control (expressionof each gene is normalized between −1.00 and 1.00 according to the colorscale). mRNAs preferentially upregulated (red) or downregulated(green) >2-fold are shown. (C) Number of mRNAs upregulated (open bars)or downregulated (solid bars) after stimulation with indicatedcytokines. IL-2, H9, H9-RET, and H9-RETR respectively induced 731, 742,65, and 23 mRNAs and repressed 437, 397, 13, and 46 mRNAs (foldchange >2; p-value <1e-10). (D) Number of ChIP-Seq-based STAT5B bindingsites and their genome-wide distribution in pre-activated CD8⁺ T cellstreated with the IL-2 variants. Shown are 5′ untranslated regions (5′UTR), exons, introns, and 3′ untranslated regions (3′UTR) as defined inhuman RefSeq database (assembly GRCh37.p9). 5 kb upstream of TSS wasdesignated as the promoter region. (E) IL-2-induced STAT5B peaks wereused to perform heat map clustering centered ˜1 kb upstream and 1 kbdownstream of the STAT ‘peak summit’ (indicated by position “0”). Thestrength of binding induced by IL-2, H9, H9-RET, and H9-RETR isindicated by the intensity of red. (F) Expression of IL2RA, LTA, CISH,IL7RA, and BCL6 RNA relative to RPL7 in pre-activated cells leftunstimulated or stimulated with IL-2, H9, H9-RET, and H9-RETR for 24 hr.Data are representative of at least two experiments, except for theRNA-Seq experiment, in which select genes were confirmed by RT-PCR (seetext).

FIG. 17 depicts CD25 expression on pre-activated CD8⁺ T cells treatedwith IL-2, H9, H9-RE, H9-T, H9-RET, or H9-RETR. Data are representativeof three independent experiments.

FIG. 18A-18J shows that H9-RETR inhibits IL-2R-mediated signaling. (Aand B) H9-RET and H9-RETR are competitive inhibitors of IL-2 and IL-15.Pre-activated human CD8⁺ T cells were incubated with the indicatedconcentrations of IL-2 or IL-15 in the absence or presence of 1 μg/ml ofH9-RET or H9-RETR. (C and D) H9-RETR more potently blocks IL-2- andIL-15-induced STAT5 phosphorylation than anti-Tac or Mikβ1 mAbs inpre-activated CD8⁺ T cells. (E and F) H9-RETR more potently inhibitsIL-2-induced or IL-15-induced proliferation than anti-Tac or Mikβ1.Shown are means f S.E.M. Data are representative of three independentexperiments. (G) H9-RETR inhibits IL-2-induced (upper panels) andIL-15-induced (lower panels) STAT5 phosphorylation in vivo. C57BL6 micewere injected (i.p) with Fc4 or Fc4-H9-RETR 60 min prior to IL-2 orIL-15. pSTAT5 was measured 30 min later in splenic CD4⁺CD25⁺FoxP3⁺ Tcells. MFIs are indicated. Data are representative of three independentexperiments. (H) Fc4-H9-RETR attenuates GVHD. BALB/c mice wereirradiated (950 cGy) and transplanted with 10 million T-depleted bonemarrow (BM) cells without or with 2 million Treg-depleted pan-T cellsfrom C57BL/6 mice. Mice receiving pan-T cells were treated with Fc4 orFc4-H9-RETR fusion protein by i.p injections for 10 days with 100μg/dose twice a day. Data represent survival curves pooled from threeindependent experiments, and analyzed using the Kaplan-Meier method andthe log-rank test. p=0.0001 for Fc4 vs H9-RETR-Fc4. (I) Blocking ofproliferation of ED40515(+) T cells cultured with 50 U/ml IL-2 for 3days in the presence or absence of UPC10, daclizumab, Mikβ1, or H9-RETR,as indicated. Data are representative of two experiments, each performedin triplicate. (J) Spontaneous six day proliferation assay of cells froma patient with smoldering ATL, treated with UPC10, daclizumab, Mikβ1, orH9-RETR. Assays were performed in triplicate.

FIG. 19A-19C shows the effects of IL-2 muteins H9-RET and h9-RETR onCD8+ T cells. (A) H9-RET and H9-RETR inhibited IL-2-induced CD25expression on pre-activated CD8⁺ T cells. (B) IL2RA mRNA levels(normalized to RPL7 expression) in preactivated human CD8⁺ T cellsstimulated with IL-2 in the presence or absence of H9-RET or H9-RETR.Data are representative of three independent experiments. (C) Inhibitionof IL-2- and IL-15-induced T-cell proliferation by H9-RETR. Freshlyisolated CD8⁺ T cell were CFSE-labeled and stimulated with IL-2 or IL-15(1 μg/ml) in the presence or absence of 1 μg/ml H9-RET or H9-RETR, andCFSE dilution assessed. Data are representative of three independentexperiments (A and C) or are pooled from two independent experimentsdone in triplicate (B).

FIG. 20A-20B shows the effects of IL-2 muteins H9-RET and h9-RETR onCD8+ T cells. (A) Both H9-RET and H9-RETR inhibit TCR-inducedproliferation of freshly isolated CD8⁺ T cells. Cells were labeled withCFSE, stimulated with plate bound anti-CD3 (2 μg/ml)+ soluble anti-CD28(1 μg/ml) for 4 days with or without 1 μg/ml of H9-RET or H9-RETR, andCFSE dilution assessed by flow cytometry. (B) 1 μg/ml of either H9-RETor H9-RETR inhibited TCR-induced CD25 expression on peripheral bloodCD8⁺ T cells stimulated for 4 days with 2 μg/ml anti-CD3+1 μg/mlanti-CD28. Data are representative of three independent experiments.

FIG. 21 shows that H9-RET and H9-RETR block Th1, Th9, and Tregdifferentiation but promote Th17 differentiation. Cells weredifferentiated under various T-helper polarizing conditions in theabsence or presence of H9-RET or H9-RETR. Data are representative of atleast two independent experiments for each type of cell.

FIG. 22A-22C shows that H9-RETR blocks IL-2-induced NK cell activationand cytotoxicity. (A) Unlike IL-2, neither H9-RET nor H9-RETR (1 μg/ml)stimulated CD69 expression in primary human NK cells after incubationfor 24 h, but H9-RETR inhibited IL-2 (100 ng/ml)-induced CD69expression. The experiment was performed twice. (B, C) Neither H9-RETnor H9-RETR stimulated cytotoxicity in primary human NK cells, andH9-RETR at 10³ ng/mL inhibited IL-2-induced NK cell cytotoxicity ofHER18 (B) and K562 (C) target cells. NK cells and target cells wereincubated at a 10:1 ratio for 4 h in the presence of the indicatedcytokines. HER18 cell lysis was determined by ⁵¹Cr release and performedin triplicate; lysis of K562 cells was assessed by flow cytometry. Fourindependent experiments were performed.

FIG. 23 is a schematic of the protocol used for the experiment in FIG.18G.

DETAILED DESCRIPTION

In order for the present disclosure to be more readily understood,certain terms and phrases are defined below as well as throughout thespecification.

Definitions

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, AdvancedOrganic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley& Sons (New York, N.Y. 2001); and Sambrook and Russell, MolecularCloning. A Laboratory Manual 3rd ed., Cold Spring harbor LaboratoryPress (Cold Spring Harbor, N.Y. 2001), provide one skilled in the artwith a general guide to many terms used in the present disclosure. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

As used herein, “IL-2” means wild-type IL-2, whether native orrecombinant. Mature human IL-2 occurs as a 133 amino acid sequence (lessthe signal peptide, consisting of an additional 20 N-terminal aminoacids), as described in Fujita, et. al., PNAS USA, 80, 7437-7441 (1983).The amino acid sequence of human IL-2 (SEQ ID NO: 1) is found in Genbankunder accession locator NP_000577.2. The amino acid sequence of maturehuman IL-2 is depicted in SEQ ID NO: 2. The murine (Mus musculus) IL-2amino acid sequence is found in Genbank under accession locator (SEQ IDNO: 3). The amino acid sequence of mature murine IL-2 is depicted in SEQID NO: 4.

SEQ ID NO: 1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS IISTLT SEQ ID NO: 2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 3MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESA TVVDFLRRWIAFCQSIISTSPQSEQ ID NO: 4 APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTS PQ

As used herein, “IL-2 mutein” means an IL-2 polypeptide wherein specificsubstitutions to the interleukin-2 protein have been made. The IL-2muteins are characterized by amino acid insertions, deletions,substitutions and modifications at one or more sites in or at the otherresidues of the native IL-2 polypeptide chain. In accordance with thisdisclosure, any such insertions, deletions, substitutions andmodifications result in an IL-2 mutein that retains the IL-2Rβ bindingactivity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more amino acids.

Muteins also include conservative modifications and substitutions atother positions of IL-2 (i.e., those that have a minimal effect on thesecondary or tertiary structure of the mutein). Such conservativesubstitutions include those described by Dayhoff in The Atlas of ProteinSequence and Structure 5 (1978), and by Argos in EMBO J., 8:779-785(1989). For example, amino acids belonging to one of the followinggroups represent conservative changes: Group I: ala, pro, gly, gln, asn,ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met,ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; andGroup VI: asp, glu.

“Numbered in accordance with IL-2” means identifying a chosen amino acidwith reference to the position at which that amino acid normally occursin the mature sequence of wild type IL-2, for example R81 refers to theeighty-first amino acid, arginine, that occurs in SEQ ID NO: 2.

The term “cell types having the IL-2Rαβγ receptor” means the cells knownto have this receptor type, i.e., T cells, activated T cells, B cells,activated monocytes, and activated NK cells. The term “cell types havingthe IL-2Rβγ receptor” means the cells known to have that receptor type,i.e., B cells, resting monocytes, and resting NK cells.

The term “identity,” as used herein in reference to polypeptide or DNAsequences, refers to the subunit sequence identity between twomolecules. When a subunit position in both of the molecules is occupiedby the same monomeric subunit (i.e., the same amino acid residue ornucleotide), then the molecules are identical at that position. Thesimilarity between two amino acid or two nucleotide sequences is adirect function of the number of identical positions. In general, thesequences are aligned so that the highest order match is obtained. Ifnecessary, identity can be calculated using published techniques andwidely available computer programs, such as the GCS program package(Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN,FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequenceidentity can be measured using sequence analysis software such as theSequence Analysis Software Package of the Genetics Computer Group at theUniversity of Wisconsin Biotechnology Center (1710 University Avenue,Madison, Wis. 53705), with the default parameters thereof.

The terms “polypeptide,” “protein” or “peptide” refer to any chain ofamino acid residues, regardless of its length or post-translationalmodification (e.g., glycosylation or phosphorylation).

In the event the mutant IL-2 polypeptides of the disclosure are“substantially pure,” they can be at least about 60% by weight (dryweight) the polypeptide of interest, for example, a polypeptidecontaining the mutant IL-2 amino acid sequence. For example, thepolypeptide can be at least about 75%, about 80%, about 85%, about 90%,about 95% or about 99%, by weight, the polypeptide of interest. Puritycan be measured by any appropriate standard method, for example, columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

An “agonist” is a compound that interacts with a target to cause orpromote an increase in the activation of the target.

A “partial agonist” is a compound that interacts with the same target asan agonist but does not produce as great a magnitude of a biochemicaland/or physiological effect as the agonist, even by increasing thedosage of the partial agonist.

A “super agonist” is a type of agonist that is capable of producing amaximal response greater than the endogenous agonist for the targetreceptor, and thus has an efficacy of more than 100%.

An “antagonist” is a compound that opposes the actions of an agonist,e.g. by preventing, reducing, inhibiting, or neutralizing the activityof an agonist. An “antagonist” can also prevent, inhibit, or reduceconstitutive activity of a target, e.g., a target receptor, even wherethere is no identified agonist.

“Operably linked” is intended to mean that the nucleotide sequence ofinterest (i.e., a sequence encoding an IL-2 mutein) is linked to theregulatory sequence(s) in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). “Regulatory sequences” include promoters, enhancers, and otherexpression control elements (e.g., polyadenylation signals). See, forexample, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionconstructs of the invention can be introduced into host cells to therebyproduce the human IL-2 muteins disclosed herein or to producebiologically active variants thereof.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but also to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell but are still included within the scope of the term as used herein.

As used herein, the terms “transformation” and “transfection” refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, particle gun, or electroporation.

As used herein, the term “pharmaceutically acceptable carrier” includes,but is not limited to, saline, solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Supplementary active compounds (e.g., antibiotics) can also beincorporated into the compositions.

As used herein, the terms “cancer” (or “cancerous”),“hyperproliferative,” and “neoplastic” to refer to cells having thecapacity for autonomous growth (i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth). Hyperproliferativeand neoplastic disease states may be categorized as pathologic (i.e.,characterizing or constituting a disease state), or they may becategorized as non-pathologic (i.e., as a deviation from normal but notassociated with a disease state). The terms are meant to include alltypes of cancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. “Pathologichyperproliferative” cells occur in disease states characterized bymalignant tumor growth. Examples of non-pathologic hyperproliferativecells include proliferation of cells associated with wound repair. Theterms “cancer” or “neoplasm” are used to refer to malignancies of thevarious organ systems, including those affecting the lung, breast,thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, andthe genitourinary tract, as well as to adenocarcinomas which aregenerally considered to include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

The term “carcinoma” is art-recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. An “adenocarcinoma” refersto a carcinoma derived from glandular tissue or in which the tumor cellsform recognizable glandular structures.

As used herein, the term “hematopoietic neoplastic disorders” refers todiseases involving hyperplastic/neoplastic cells of hematopoieticorigin, e.g., arising from myeloid, lymphoid or erythroid lineages, orprecursor cells thereof. Preferably, the diseases arise from poorlydifferentiated acute leukemias (e.g., erythroblastic leukemia and acutemegakaryoblastic leukemia). Additional exemplary myeloid disordersinclude, but are not limited to, acute promyeloid leukemia (APML), acutemyelogenous leukemia (AML) and chronic myelogenous leukemia (CML)(reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97);lymphoid malignancies include, but are not limited to acutelymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional forms of malignant lymphomas include, but are not limited tonon-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Stemberg disease.

IL-2 Muteins IL-2 Mutein Partial Agonists and Antagonists

In one aspect, provided herein are IL-2 muteins that are partialagonists and antagonists. In certain embodiments, provided herein areIL-2 muteins that contain one or more mutations that reduces the bindingaffinity of the IL-2 mutein for IL-2Rγ_(c) receptor as compared towild-type IL-2 (e.g., human IL-2, SEQ ID NO: 2). As used herein, theterms, “common gamma chain, “γ_(c),” IL-2Rγ_(c),” “Yc,” “IL-2Rγ,” “IL-2receptor subunit gamma,” and “IL-2RG” (Genbank accession numbers:NM_000206 and NP_000197 (human) and NM_013563 and NP_038591 (mouse)) allrefer to a member of the type I cytokine receptor family that is acytokine receptor subunit to the receptor complexes for at least sixdifferent interleukin receptor including, but not limited to, IL-2,IL-4, IL-7, IL-9, IL-15, and IL-21 receptors. IL-2R γ_(c) interacts withIL-2Rβ to form an intermediate affinity IL-2 receptor primarily onmemory T cells and natural killer (NK) cells and interacts with IL-2Rαand IL-2Rβ to form a high affinity IL-2 receptor on activated T cellsand regulator T cells (Tregs). Without being bound by any particulartheory of operation, it is believed that such muteins can function asIL-2 partial agonists or antagonists by attenuating or inhibitingIL-2β/IL-2γ_(c) heterodimerization and signaling upon binding to IL-2Rβon IL-2Rβ+/IL-2Rγ_(c)+ cells (e.g., resting T cells and natural killer(NK) cells.

Exemplary subject IL-2 muteins are at least about 50%, at least about65%, at least about 70%, at least about 80%, at least about 85%, atleast about 87%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% identical to wild-type IL-2. The mutation can consist of achange in the number or content of amino acid residues. For example, themutant IL-2 can have a greater or a lesser number of amino acid residuesthan wild-type IL-2. Alternatively, or in addition, an exemplary mutantpolypeptide can contain a substitution of one or more amino acidresidues that are present in the wild-type IL-2. In various embodiments,the mutant IL-2 polypeptide can differ from wild-type IL-2 by theaddition, deletion, or substitution of a single amino acid residue.

By way of illustration, an IL-2 mutein that includes an amino acidsequence that is at least 95% identical to the reference amino acidsequence SEQ ID NO:2 is a polypeptide that includes a sequence that isidentical to the reference sequence except for the inclusion of up tofive alterations of the reference amino acid sequence of SEQ ID NO: 2.For example, up to 5% of the amino acid residues in the referencesequence may be deleted or substituted with another amino acid, or anumber of amino acids up to 5% of the total amino acid residues in thereference sequence may be inserted into the reference sequence. Thesealterations of the reference sequence can occur at the amino (N-) orcarboxy (C-) terminal positions of the reference amino acid sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

In certain embodiments, the IL-2 mutein binds IL-2Rγ_(c) with anaffinity that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% less than wild-type IL-2. The binding affinity of IL-2mutein can also be expressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50,100, 200, 250 or more fold lower affinity for the IL-2γ_(c) thanwild-type IL-2. The binding affinity of a subject IL-2 mutein forIL-2γ_(c) can be measured using any suitable method known in the art.Suitable methods for measuring IL-2Rγ_(c) binding, include, but are notlimited to, radioactive ligand binding assays (e.g., saturation binding,scatchard plot, nonlinear curve fitting programs and competition bindingassays); non-radioactive ligand binding assays (e.g., fluorescencepolarization (FP), fluorescence resonance energy transfer (FRET) andsurface plasmon resonance assays (see, e.g., Drescher et al., MethodsMol Biol 493:323-343 (2009)); liquid phase ligand binding assays (e.g.,real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation);and solid phase ligand binding assays (e.g., multiwell plate assays,on-bead ligand binding assays, on-column ligand binding assays, andfilter assays).

In certain embodiments, the IL-2 mutein disrupts the association of theIL-2Rβ with the IL-2Rγ_(c) such that this IL-2Rβ/IL-2Rγ_(c) interactionis reduced by about 2%, about 5%, about 10%, about 15%, about 20%, about50%, about 75%, about 90%, about 95% or more relative to wild-type IL-2.

In some embodiments, the one or more mutations reducing the bindingaffinity of the IL-2 mutein for IL-2Rγ_(c) receptor is an amino acidsubstitution. In some embodiments, the subject IL-2 mutein consists of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acidsubstitutions as compared to a wild type IL-2 (SEQ ID NO:2). Thesubstituted amino acid residue(s) can be, but are not necessarily,conservative substitutions, which typically include substitutions withinthe following groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. In particularembodiments, the substitutions are at amino acid residues of IL-2 thatcontact the IL-2Rγ_(c) binding interface.

In certain embodiments, the amino acid substitutions are substitutionsat one or more amino acid positions of wild type IL-2 selected frompositions: 18, 22, 126, and/or 130, numbered in accordance withwild-type hIL-2 (e.g., SEQ ID NO: 2). In certain embodiments, the aminoacid substitutions that decrease IL-2R γ_(c) receptor binding affinityinclude amino acid substitutions L18R, Q22E, A126T and/or S130R orcombinations thereof.

In some embodiments, the amino acid substitution that decreases IL-2Rγ_(c) receptor binding affinity includes Q126T. In other embodiments,the amino acid substitutions that decrease IL-2R γ_(c) receptor bindingaffinity include L18R and Q22E. In some embodiments, the amino acidsubstitutions that decrease IL-2R γ_(c) receptor binding affinityinclude L18R, Q22E, and Q126T. In other embodiments, the amino acidsubstitutions that decrease IL-2R γ_(c) receptor binding affinityinclude L18R, Q22E, Q126T and S130R.

In some embodiments, the IL-2 mutein having a reduced binding affinityfor IL-2Rβ receptor further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore mutations that increase IL-2Rβ binding affinity. As used herein,the terms “IL-2Rβ” and “CD122” (Genbank accession number NM_000878 andNP_000869 (human)) both refer to a member of the type I cytokinereceptor family that interacts with IL-2Rγ_(c) to form an intermediateaffinity IL-2 receptor primarily on memory T cells and natural killer(NK) cells and interacts with IL-2Rα and IL-2Rγ_(c) to form a highaffinity IL-2 receptor on activated T cells and regulator T cells(Tregs). Without being bound by any particular theory of operation, itis believed that such IL-2 muteins that have a strong binding affinityIL-2Rβ and a weak binding affinity for IL-2Rγ_(c) serve as adominant-negative scaffold to create a “receptor signaling clamp” toblock endogenous signaling. Such IL-2 muteins would attenuateIL-2Rβ-γ_(c) heterodimerization and represent a new class ofmechanism-based IL-2 partial agonists and non-signaling (neutral)molecules that functionally act as antagonists by blocking endogenouscytokines and exerting no action of their own (see schematic in FIG. 1).

In certain embodiments, the subject IL-2 mutein includes at least onemutation (e.g., a deletion, addition, or substitution of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more aminoacid residues) relative to a wild-type IL-2 (e.g., SEQ ID NO:2), andbinds the IL-2Rβ with higher affinity than a wild-type IL-2.

In certain embodiments, the IL-2 mutein binds IL-2Rβ with an affinitythat is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% greater than wild-type IL-2. The binding affinity of IL-2 mutein canalso be expressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200,250 or more fold greater affinity for the IL-2Rβ than wild-type IL-2.Binding of the subject IL-2 mutein to IL-2Rβ can be assessed by anysuitable method known to those in the art, including, but not limited tothe methods described above.

In some embodiments, the at least one mutations increasing IL-2Rβbinding affinity is an amino acid substitution. In some embodiments, theamino acid substitutions that increase IL-2Rβ binding affinity includesubstitutions at amino acid positions 124, P65, Q74, L80, R81, L85, 186,189, 192, and/or V93 numbered in accordance with wild-type hIL-2 (SEQ IDNO: 2): In certain embodiments, the substitutions include I24V, P65H,Q74R, Q74H, Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V,I92F, and/or V93I or combinations thereof. In certain embodiments, thesubstitutions include Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V,I86V, I89V, and/or I93V or combinations thereof.

In some embodiments, the amino acid substitutions increasing IL-2Rβbinding affinity include: L80F, R81D, L85V, I86V and I92F. In someembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity include: Q74N, L80F, R81D, L85V, I86V, I89V, and I92F. In someembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity include: Q74N, L80V, R81T, L85V, I86V, and I92F. In someembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity include: Q74H, L80F, R81D, L85V, I86V and I92F. In certainembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity include: Q74S, L80F, R81D, L85V, I86V and I92F. In certainembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity include: Q74N, L80F, R81D, L85V, I86V and I92F. In someembodiments, the amino acid substitutions that increase IL-2Rβ bindingaffinity include: Q74S, R81T, L85V, and 192F.

In certain embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L80F, R81D, L85V, I86V, I92F and Q126T. In certainembodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 5) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In various embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, L80F, R81D, L85V, I86V and I92F. In certainembodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 6) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In exemplary embodiments, the subject IL-2 mutein having a greaterbinding affinity for IL-2Rβ and a reduced binding affinity for IL-2Rγ_(c) receptor as compared to wild-type human IL-2, includes the aminoacid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T.In certain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 7) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, andS130R. In certain embodiments, the IL-2 mutein has the amino acidsequence:

(SEQ ID NO: 8) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SII R TLT.

In certain embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions Q74N, L80F, R81D, L85V, I86V, I89V, I92F and Q126T. Incertain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 9) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SN V NV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In various embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74N, L80F, R81D, L85V, I86V, I89V, and I92F.In certain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 10) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SN V NV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In an exemplary embodiment, the subject IL-2 mutein having a greaterbinding affinity for IL-2Rβ and a reduced binding affinity for IL-2Rγ_(c) receptor as compared to wild-type human IL-2, includes the aminoacid substitutions L18R, Q22E, Q74N, L80F, R81D, L85V, I86V, I89V, I92F,and Q126T. In certain embodiments, the IL-2 mutein has the amino acidsequence:

(SEQ ID NO: 11) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SN V NV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74N, L80F, R81D, L85V, I86V, I89V, I92F,Q126T, and S130R. In certain embodiments, the IL-2 mutein has the aminoacid sequence:

(SEQ ID NO: 12) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SN V NV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SII R TLT.

In certain embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions Q74N, L80V, R81T, L85V, 186V, 192F and Q126T. In certainembodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 13) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH VT PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In various embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74N, L80V, R81T, L85V, 186V and 192F. Incertain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 14) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH VT PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In various embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74N, L80V, R81T, L85V, I86V, I92F, and Q126T.In certain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 15) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH VT PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74N, L80V, R81T, L85V, I86V, I92F, Q126T, andS130R. In certain embodiments, the IL-2 mutein has the amino acidsequence:

(SEQ ID NO: 16) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH VT PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SII R TLT.

In certain embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions Q74H, L80F, R81D, L85V, I86V, I92F and Q126T. In certainembodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 17) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA H SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In various embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74H, L80F, R81D, L85V, 186V and I92F. Incertain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 18) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA H SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In an exemplary embodiment, the subject IL-2 mutein having a greaterbinding affinity for IL-2Rβ and a reduced binding affinity for IL-2Rγ_(c) receptor as compared to wild-type human IL-2, includes the aminoacid substitutions L18R, Q22E, Q74H, L80F, R81D, L85V, I86V, I92F, andQ126T. In certain embodiments, the IL-2 mutein has the amino acidsequence:

(SEQ ID NO: 19) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA H SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74H, L80F, R81D, L85V, I86V, I92F, Q126T, andS130R. In certain embodiments, the IL-2 mutein has the amino acidsequence:

(SEQ ID NO: 20) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA H SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SII R TLT.

In certain embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions Q74S, L80F, R81D, L85V, 186V, 192F and Q126T. In certainembodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 21) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74S, L80F, R81D, L85V, I86V and I92F. Incertain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 22) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In various embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74S, L80F, R81D, L85V, I86V, I92F, and Q126T.In certain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 23) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74S, L80F, R81D, L85V, I86V, I92F, Q126T, andS130R. In certain embodiments, the IL-2 mutein has the amino acidsequence:

(SEQ ID NO: 24) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SII R TLT.

In certain embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions Q74N, L80F, R81D, L85V, 186V, 192F and Q126T. In certainembodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 25) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74N, L80F, R81D, L85V, I86V and I92F. Incertain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 26) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In various embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74N, L80F, R81D, L85V, I86V, I92F, and Q126T.In certain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 27) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74N, L80F, R81D, L85V, I86V, I92F, Q126T, andS130R. In certain embodiments, the IL-2 mutein has the amino acidsequence:

(SEQ ID NO: 28) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SII R TLT.

In certain embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions Q74S, R81T, L85V, I92F and Q126T. In certain embodiments,the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 29) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFHL T PRD V ISNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22E, Q74S, R81T, L85V, and I92F. In certainembodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 30) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFHL T PRD V ISNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In certain embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22S, Q74H, R81T, L85V, I92F, and Q126T. In certainembodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 31) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFHL T PRD V ISNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SIISTLT.

In some embodiments, the subject IL-2 mutein having a greater bindingaffinity for IL-2Rβ and a reduced binding affinity for IL-2R γ_(c)receptor as compared to wild-type human IL-2, includes the amino acidsubstitutions L18R, Q22S, Q74H, R81T, L85V, I92F, Q126T, and S130R. Incertain embodiments, the IL-2 mutein has the amino acid sequence:

(SEQ ID NO: 32) APTSSSTKKTQLQLEHL R LDL E MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFHL T PRD V ISNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFC T SII R TLT.

In various embodiments, the subject IL-2 mutein has an amino acidsequence according to the formula:

A-P-T-S-S-S-T-K-K-T-Q-L-Q-L-E-H-L-(X₁)_(n)-L-D-L-(X²)_(n)-−M-(X³)_(n)-−L-N-G-I-N-N-Y-K-N-P-K-L-T-R-M-L-T-F-K-F-Y-M-P-K-K-A-T-E-L-K-H-L-Q-C-L-E-E-E-L-K-(X⁴)_(n)-⁻L-E-E-V-L-N-L-A-(X⁵)_(n)-⁻S-K-N-F-H-(X⁶)_(n)-(X⁷)_(n)-−P-R-D-(X⁸)_(n)-−(X⁹)_(n)-−S-N-(X¹⁰)_(n)-−N-V-(X¹¹)_(n)-−(X¹²)_(n)-−L-E-L-K-G-S-E-T-T-F-M-C-E-Y-A-D-E-T-A-T-I-V-E-F-L-N-RW-I-T-F-C-(X¹³)_(n)-−S-I-I-(X¹⁴)_(n)-−T-L-T,wherein:

each n is individually selected from 0 or 1;

X¹ is L (wild-type) or R;

X² is Q (wild-type) or E;

X³ is I (wild-type) or V;

X⁴ is P (wild-type) or H;

X⁵ is Q (wild-type), R, H, N or S;

X⁶ is L (wild-type), F or V;

X⁷ is R (wild-type), I, T or D;

X⁸ is L (wild-type) or V;

X⁹ is I (wild-type) or V;

X¹⁰ is I (wild-type) or V;

X¹¹ is I (wild-type) or F;

X¹² is V (wild-type) or I;

X¹³ is A (wild-type) or T; and

X¹⁴ is S (wild-type) or R. (SEQ ID NO: 50).

In certain embodiments of the IL-2 mutein according to SEQ ID NO: 50, anamino acid at least at one of X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰,X¹¹, X¹², X¹³ or X¹⁴ is not a wild-type amino acid. In some embodiments,an amino acid at least at two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, or fourteen of X¹, X², X³, X⁴, X⁵,X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³ or X¹⁴ is not a wild-type amino acid.In some embodiments, the IL-2 mutein has at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99% orabout 100% homology with the IL-2 mutein of SEQ ID NO: 50.

In some embodiments, the subject IL-2 muteins that are partial agonistshave one or more reduced functions as compared to wild-type IL-2.

In certain embodiments, the IL-2 mutein has reduced capabilities tostimulate one or more signaling pathways that are dependent onIL-2Rβ/IL-2Rγ_(c) heterodimerization. In some embodiments, the subjectIL-2 mutein has a reduced capability to stimulate STAT5 phosphorylationin an IL-2Rβ+ cell as compared to wild-type hIL-2. In some embodiments,the IL-2 mutein stimulates STAT5 phosphorylation in an IL-2Rβ+ cell at alevel that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less of the level thatwild-type IL-2 stimulates STAT5 phosphorylation in the same cell. Insome embodiments, the IL-2Rβ+ cell is a T cell. In particularembodiments, the T cell is a CD8+ T cell. In some embodiments, the CD8+T cell is a freshly isolated CD8+ T cell. In other embodiments, the CD8+T cell T cell is an activated CD8+ T cell. In other embodiments, theIL-2Rβ+ cell is a natural killer (NK) cell.

In some embodiments, the mutein has a reduced capability to stimulateERK1/ERK2 signaling in an IL-2Rβ+ cell as compared to wild-type hIL-2.In some embodiments, the IL-2 mutein stimulates pERK1/ERK2 signaling inan IL-2Rβ+ cell at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less ofthe level that wild-type IL-2 stimulates pERK1/ERK2 signaling in thesame cell. In some embodiments, the IL-2Rβ+ cell is a T cell. Inparticular embodiments, the T cell is a CD8+ T cell. In someembodiments, the CD8+ T cell is a freshly isolated CD8+ T cell. In otherembodiments, the CD8+ T cell T cell is an activated CD8+ T cell. Inother embodiments, the IL-2Rβ+ cell is a natural killer (NK) cell.

STAT5 and ERK1/2 signaling can be measure, for example, byphosphorylation of STAT5 and ERK1/2 using any suitable method known inthe art. For example, STAT5 and ERK1/2 phosphorylation can be measuredusing antibodies specific for the phosphorylated version of thesemolecules in combination with flow cytometry analysis as describedherein.

In some embodiments, the mutein has a reduced capability to stimulate PI3-kinase signaling in a IL-2Rβ+ cell as compared to wild-type hIL-2. Insome embodiments, the IL-2 mutein stimulates PI 3-kinase signaling in anIL-2Rβ+ cell at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less ofthe level that wild-type IL-2 stimulates PI 3-kinase signaling in thesame cell. In some embodiments, the IL-2Rβ+ cell is a T cell. Inparticular embodiments, the T cell is a CD8+ T cell. In someembodiments, the CD8+ T cell T cell is an activated CD8+ T cell. Inother embodiments, the IL-2Rβ+ cell is a natural killer (NK) cell.

PI 3-kinase signaling can be measured using any suitable method known inthe art. For example, PI 3-kinase signaling can be measured usingantibodies that are specific for phospho-S6 ribosomal protein inconjunction with flow cytometry analysis as described herein.

In certain embodiments, the mutein has a reduced capability to inducelymphocyte proliferation as compared to wild-type IL-2. In someembodiments, the lymphocyte is a T cell. In particular embodiments, thelymphocyte is a primary CD8+ T cell. In other embodiments, thelymphocyte is an activated CD8+ T cell. Cell proliferation can bemeasured using any suitable method known in the art. For example,lymphocyte proliferation can be measured using a carboxyfluoresceindiacetate succinimidyul diester (CFSE) dilution assay or by[³H]-thymidine incorporation, as described herein. In some embodiments,the IL-2 mutein induce lymphocyte proliferation at a level that is 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or less of the level that wild-type IL-2 inducelymphocyte proliferation.

In some embodiments, the IL-2 mutein has a reduced capability toactivate IL-2Rα expression in a lymphocyte as compared to wild-typeIL-2. In some embodiments, the IL-2 mutein activates IL-2Rα expressionin a lymphocyte at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less ofthe level that wild-type IL-2 activates IL-2Rα expression in the samecell. In some embodiments, the lymphocyte is a CD8+ T cell. In someembodiments, the CD8+ T cell is a freshly isolated CD8+ T cell. In otherembodiments, the CD8+ T cell is an activated CD8+ T cell.

Without being bound by any particular theory of operation, it isbelieved that IL-2 muteins that exhibit enhanced IL-2Rβ binding anddecreased IL-2Rγ_(c) can function as dominant negative IL-2 antagonistsand interfere with one or more IL-2 dependent functions. Suchantagonists function by interfering with IL-2 binding to IL-2Rβ, whilealso inhibiting IL-2Rβ/IL-2Rγ_(c) heterodimerization. Moreover, sinceIL-15 signaling functions through IL-2Rβ/IL-2Rγ_(c) receptor binding, itis believed that such IL-2 muteins can also function as IL-15antagonists. In certain embodiments, the IL-2 mutein inhibits one ormore IL-2 and/or IL-15 function.

In some embodiments, the IL-2 mutein that inhibits one or more IL-2and/or IL-15 functions includes an amino acid substitution at amino acidpositions 18, 22, and 126, numbered in accordance with wild-type hIL-2.In some embodiments, the amino acid substitutions of the IL-2 muteininclude L18R, Q22E, and Q126T, numbered in accordance with wild-typehIL-2.

In some embodiments, the IL-2 mutein that inhibits one or more IL-2and/or IL-15 functions includes an amino acid substitution at amino acidpositions 18, 22, 126, and 130 numbered in accordance with wild-typehIL-2. In some embodiments, the amino acid substitutions of the IL-2mutein include L18R, Q22E, Q126T and S130R, numbered in accordance withwild-type hIL-2.

In certain embodiments the mutein is an inhibitor of IL-2 and/or IL-15STAT5 phosphorylation in CD8+ T cells. In some embodiments, the muteinis an inhibitor of IL-2 and/or IL-15 induced proliferation of CD8+ Tcells. In some embodiments, the mutein is an inhibitor of IL-2dependent, TCR-induced cell proliferation.

IL-2 promotes Th1, Th9, and Treg T cell differentiation and inhibitsTh17 differentiation. Therefore, without being bound by any particulartheory of operation, it is believed that IL-2 muteins that function asIL-2 antagonists are capable of inhibiting Th1, Th9, and/or Treg celldifferentiation or promoting Th17 cell differentiation. In someembodiments, the IL-2 mutein is an inhibitor of IL-2 dependent Th1, Th9and/or Treg differentiation. In certain embodiments, the mutein is apromoter of Th17 differentiation.

In certain embodiments the mutein is an inhibitor an inhibitor of IL-2dependent activation of natural killer (NK) cells. IL-2 activation of NKcells can be measured by any suitable method known in the art, forexample, by measuring IL-2 induced CD69 expression and/or cytotoxicity,as described herein.

Recombinant Expression of IL-2 Muteins, Expression Vectors and HostCells

In various embodiments, polypeptides used in the practice of the instantinvention are synthetic, or are produced by expression of a recombinantnucleic acid molecule. In the event the polypeptide is a chimera (e.g.,a fusion protein containing at least a mutant IL-2 polypeptide and aheterologous polypeptide), it can be encoded by a hybrid nucleic acidmolecule containing one sequence that encodes all or part of the mutantIL-2, and a second sequence that encodes all or part of the heterologouspolypeptide. For example, subject IL-2 muteins described herein may befused to a hexa-histidine tag to facilitate purification of bacteriallyexpressed protein, or to a hemagglutinin tag to facilitate purificationof protein expressed in eukaryotic cells.

Methods for constructing a DNA sequence encoding the IL-2 muteins andexpressing those sequences in a suitably transformed host include, butare not limited to, using a PCR-assisted mutagenesis technique.Mutations that consist of deletions or additions of amino acid residuesto an IL-2 polypeptide can also be made with standard recombinanttechniques. In the event of a deletion or addition, the nucleic acidmolecule encoding IL-2 is optionally digested with an appropriaterestriction endonuclease. The resulting fragment can either be expresseddirectly or manipulated further by, for example, ligating it to a secondfragment. The ligation may be facilitated if the two ends of the nucleicacid molecules contain complementary nucleotides that overlap oneanother, but blunt-ended fragments can also be ligated. PCR-generatednucleic acids can also be used to generate various mutant sequences.

The complete amino acid sequence can be used to construct aback-translated gene. A DNA oligomer containing a nucleotide sequencecoding for IL-2 mutein can be synthesized. For example, several smalloligonucleotides coding for portions of the desired polypeptide can besynthesized and then ligated. The individual oligonucleotides typicallycontain 5′ or 3′ overhangs for complementary assembly.

In addition to generating mutant polypeptides via expression of nucleicacid molecules that have been altered by recombinant molecularbiological techniques, subject IL-2 muteins can be chemicallysynthesized. Chemically synthesized polypeptides are routinely generatedby those of skill in the art.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the DNA sequences encoding an IL-2 mutein will be inserted intoan expression vector and operatively linked to an expression controlsequence appropriate for expression of the IL-2 mutein in the desiredtransformed host. Proper assembly can be confirmed by nucleotidesequencing, restriction mapping, and expression of a biologically activepolypeptide in a suitable host. As is well known in the art, in order toobtain high expression levels of a transfected gene in a host, the genemust be operatively linked to transcriptional and translationalexpression control sequences that are functional in the chosenexpression host.

The DNA sequence encoding the IL-2 mutein, whether prepared by sitedirected mutagenesis, chemical synthesis or other methods, can alsoinclude DNA sequences that encode a signal sequence. Such signalsequence, if present, should be one recognized by the cell chosen forexpression of the IL-2 mutein. It can be prokaryotic, eukaryotic or acombination of the two. It can also be the signal sequence of nativeIL-2. The inclusion of a signal sequence depends on whether it isdesired to secrete the IL-2 mutein from the recombinant cells in whichit is made. If the chosen cells are prokaryotic, it generally ispreferred that the DNA sequence not encode a signal sequence. If thechosen cells are eukaryotic, it generally is preferred that a signalsequence be encoded and most preferably that the wild-type IL-2 signalsequence be used.

IL-2 Mutein Fusion Proteins

As noted above, exemplary subject IL-2 muteins can be prepared as fusionor chimeric polypeptides that include a subject IL-2 muteins and aheterologous polypeptide (i.e., a polypeptide that is not IL-2 or amutant thereof) (see, e.g., U.S. Pat. No. 6,451,308). Exemplaryheterologous polypeptides can increase the circulating half-life of thechimeric polypeptide in vivo, and may, therefore, further enhance theproperties of the mutant IL-2 polypeptides. In various embodiments, thepolypeptide that increases the circulating half-life may be a serumalbumin, such as human serum albumin, or the Fc region of the IgGsubclass of antibodies that lacks the IgG heavy chain variable region.Exemplary Fc regions can include a mutation that inhibits complementfixation and Fc receptor binding, or it may be lytic, i.e., able to bindcomplement or to lyse cells via another mechanism, such asantibody-dependent complement lysis (ADCC; U.S. Ser. No. 08/355,502filed Dec. 12, 1994).

The “Fc region” can be a naturally occurring or synthetic polypeptidethat is homologous to the IgG C-terminal domain produced by digestion ofIgG with papain. IgG Fc has a molecular weight of approximately 50 kDa.The mutant IL-2 polypeptides can include the entire Fc region, or asmaller portion that retains the ability to extend the circulatinghalf-life of a chimeric polypeptide of which it is a part. In addition,full-length or fragmented Fc regions can be variants of the wild-typemolecule. That is, they can contain mutations that may or may not affectthe function of the polypeptides; as described further below, nativeactivity is not necessary or desired in all cases. In certainembodiments, the IL-2 mutein fusion protein (e.g., an IL-2 partialagonist or antagonist as described herein) includes an IgG1, IgG2, IgG3,or IgG4 Fc region.

The Fc region can be “lytic” or “non-lytic,” but is typically non-lytic.A non-lytic Fc region typically lacks a high affinity Fc receptorbinding site and a C′1q binding site. The high affinity Fc receptorbinding site of murine IgG Fc includes the Leu residue at position 235of IgG Fc. Thus, the Fc receptor binding site can be destroyed bymutating or deleting Leu 235. For example, substitution of Glu for Leu235 inhibits the ability of the Fc region to bind the high affinity Fcreceptor. The murine C′1q binding site can be functionally destroyed bymutating or deleting the Glu 318, Lys 320, and Lys 322 residues of IgG.For example, substitution of Ala residues for Glu 318, Lys 320, and Lys322 renders IgG1 Fc unable to direct antibody-dependent complementlysis. In contrast, a lytic IgG Fc region has a high affinity Fcreceptor binding site and a C′1q binding site. The high affinity Fcreceptor binding site includes the Leu residue at position 235 of IgGFc, and the C′1q binding site includes the Glu 318, Lys 320, and Lys 322residues of IgG1. Lytic IgG Fc has wild-type residues or conservativeamino acid substitutions at these sites. Lytic IgG Fc can target cellsfor antibody dependent cellular cytotoxicity or complement directedcytolysis (CDC). Appropriate mutations for human IgG are also known(see, e.g., Morrison et al., The Immunologist 2:119-124, 1994; andBrekke et al., The Immunologist 2: 125, 1994).

In other embodiments, the chimeric polypeptide can include a subjectIL-2 mutein and a polypeptide that functions as an antigenic tag, suchas a FLAG sequence. FLAG sequences are recognized by biotinylated,highly specific, anti-FLAG antibodies, as described herein (see alsoBlanar et al., Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad.Sci. USA 89:8145, 1992). In some embodiments, the chimeric polypeptidefurther comprises a C-terminal c-myc epitope tag.

In other embodiments, the chimeric polypeptide includes the mutant IL-2polypeptide and a heterologous polypeptide that functions to enhanceexpression or direct cellular localization of the mutant IL-2polypeptide, such as the Aga2p agglutinin subunit (see, e.g., Boder andWittrup, Nature Biotechnol. 15:553-7, 1997).

In other embodiments, a chimeric polypeptide including a mutant IL-2 andan antibody or antigen-binding portion thereof can be generated. Theantibody or antigen-binding component of the chimeric protein can serveas a targeting moiety. For example, it can be used to localize thechimeric protein to a particular subset of cells or target molecule.Methods of generating cytokine-antibody chimeric polypeptides aredescribed, for example, in U.S. Pat. No. 6,617,135.

Nucleic Acid Molecules Encoding Mutant IL-2

In some embodiments the subject IL-2 mutein, either alone or as a partof a chimeric polypeptide, such as those described above, can beobtained by expression of a nucleic acid molecule. Just as IL-2 muteinscan be described in terms of their identity with wild-type IL-2polypeptides, the nucleic acid molecules encoding them will necessarilyhave a certain identity with those that encode wild-type IL-2. Forexample, the nucleic acid molecule encoding a subject IL-2 mutein can beat least 50%, at least 65%, preferably at least 75%, more preferably atleast 85%, and most preferably at least 95% (e.g., 99%) identical to thenucleic acid encoding wild-type IL-2 (e.g., SEQ ID NO:2).

The nucleic acid molecules provided can contain naturally occurringsequences, or sequences that differ from those that occur naturally,but, due to the degeneracy of the genetic code, encode the samepolypeptide. These nucleic acid molecules can consist of RNA or DNA (forexample, genomic DNA, cDNA, or synthetic DNA, such as that produced byphosphoramidite-based synthesis), or combinations or modifications ofthe nucleotides within these types of nucleic acids. In addition, thenucleic acid molecules can be double-stranded or single-stranded (i.e.,either a sense or an antisense strand).

The nucleic acid molecules are not limited to sequences that encodepolypeptides; some or all of the non-coding sequences that lie upstreamor downstream from a coding sequence (e.g., the coding sequence of IL-2)can also be included. Those of ordinary skill in the art of molecularbiology are familiar with routine procedures for isolating nucleic acidmolecules. They can, for example, be generated by treatment of genomicDNA with restriction endonucleases, or by performance of the polymerasechain reaction (PCR). In the event the nucleic acid molecule is aribonucleic acid (RNA), molecules can be produced, for example, by invitro transcription.

Exemplary isolated nucleic acid molecules of the present disclosure caninclude fragments not found as such in the natural state. Thus, thisdisclosure encompasses recombinant molecules, such as those in which anucleic acid sequence (for example, a sequence encoding a mutant IL-2)is incorporated into a vector (e.g., a plasmid or viral vector) or intothe genome of a heterologous cell (or the genome of a homologous cell,at a position other than the natural chromosomal location).

As described above, the subject IL-2 mutein may exist as a part of achimeric polypeptide. In addition to, or in place of, the heterologouspolypeptides described above, a subject nucleic acid molecule cancontain sequences encoding a “marker” or “reporter.” Examples of markeror reporter genes include β-lactamase, chloramphenicol acetyltransferase(CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase(neo^(r), G418′), dihydrofolate reductase (DHFR),hygromycin-B-hosphotransferase (HPH), thymidine kinase (TK), lacz(encoding β-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). One of skill in the art will be awareof additional useful reagents, for example, of additional sequences thatcan serve the function of a marker or reporter.

The subject nucleic acid molecules can be obtained by introducing amutation into IL-2-encoding DNA obtained from any biological cell, suchas the cell of a mammal. Thus, the subject nucleic acids (and thepolypeptides they encode) can be those of a mouse, rat, guinea pig, cow,sheep, horse, pig, rabbit, monkey, baboon, dog, or cat. In oneembodiment, the nucleic acid molecules will be those of a human.

Expression of Mutant IL-2 Gene Products

The nucleic acid molecules described above can be contained within avector that is capable of directing their expression in, for example, acell that has been transduced with the vector. Accordingly, in additionto the subject IL-2 muteins, expression vectors containing a nucleicacid molecule encoding a subject IL-2 mutein and cells transfected withthese vectors are among the preferred embodiments.

It should of course be understood that not all vectors and expressioncontrol sequences will function equally well to express the DNAsequences described herein. Neither will all hosts function equally wellwith the same expression system. However, one of skill in the art maymake a selection among these vectors, expression control sequences andhosts without undue experimentation. For example, in selecting a vector,the host must be considered because the vector must replicate in it. Thevector's copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered. For example, vectors thatcan be used include those that allow the DNA encoding the IL-2 muteinsto be amplified in copy number. Such amplifiable vectors are well knownin the art. They include, for example, vectors able to be amplified byDHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufmanand Sharp, “Construction of a Modular Dihydrafolate Reductase cDNA Gene:Analysis of Signals Utilized for Efficient Expression”, Mol. Cell.Biol., 2, pp. 1304-19 (1982)) or glutamine synthetase (“GS”)amplification (see, e.g., U.S. Pat. No. 5,122,464 and European publishedapplication 338,841).

In some embodiments, the human IL-2 muteins of the present disclosurewill be expressed from vectors, preferably expression vectors. Thevectors are useful for autonomous replication in a host cell or may beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome (e.g.,nonepisomal mammalian vectors). Expression vectors are capable ofdirecting the expression of coding sequences to which they are operablylinked. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids (vectors). However, otherforms of expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses, and adeno-associated viruses) areincluded also.

Exemplary recombinant expression vectors can include one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, operably linked to the nucleic acid sequence to beexpressed.

The expression constructs or vectors can be designed for expression ofan IL-2 mutein or variant thereof in prokaryotic or eukaryotic hostcells.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. Suitable methodsfor transforming or transfecting host cells can be found in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.) and other standard molecularbiology laboratory manuals.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters. Strategies to maximize recombinant protein expression in E.coli can be found, for example, in Gottesman (1990) in Gene ExpressionTechnology: Methods in Enzymology 185 (Academic Press, San Diego,Calif.), pp. 119-128 and Wada et al. (1992) Nucleic Acids Res.20:2111-2118. Processes for growing, harvesting, disrupting, orextracting the IL-2 mutein or variant thereof from cells aresubstantially described in, for example, U.S. Pat. Nos. 4,604,377;4,738,927; 4,656,132; 4,569,790; 4,748,234; 4,530,787; 4,572,798;4,748,234; and 4,931,543, herein incorporated by reference in theirentireties.

In some embodiments the recombinant IL-2 muteins or biologically activevariants thereof can also be made in eukaryotes, such as yeast or humancells. Suitable eukaryotic host cells include insect cells (examples ofBaculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39)); yeast cells (examples of vectorsfor expression in yeast S. cerenvisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and pPicZ (InvitrogenCorporation, San Diego, Calif.)); or mammalian cells (mammalianexpression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cellsinclude Chinese hamster ovary cells (CHO) or COS cells. In mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2^(nd) ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See, Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.).

The sequences encoding the human IL-2 muteins of the present disclosurecan be optimized for expression in the host cell of interest. The G-Ccontent of the sequence can be adjusted to levels average for a givencellular host, as calculated by reference to known genes expressed inthe host cell. Methods for codon optimization are well known in the art.Codons within the IL-2 mutein coding sequence can be optimized toenhance expression in the host cell, such that about 1%, about 5%, about10%, about 25%, about 50%, about 75%, or up to 100% of the codons withinthe coding sequence have been optimized for expression in a particularhost cell.

Vectors suitable for use include T7-based vectors for use in bacteria(see, for example, Rosenberg et al., Gene 56:125, 1987), the pMSXNDexpression vector for use in mammalian cells (Lee and Nathans, J. Biol.Chem. 263:3521, 1988), and baculovirus-derived vectors (for example, theexpression vector pBacPAK9 from Clontech, Palo Alto, Calif.) for use ininsect cells.

In some embodiments nucleic acid inserts, which encode the subject IL-2muteins in such vectors, can be operably linked to a promoter, which isselected based on, for example, the cell type in which expression issought.

In selecting an expression control sequence, a variety of factors shouldalso be considered. These include, for example, the relative strength ofthe sequence, its controllability, and its compatibility with the actualDNA sequence encoding the subject IL-2 mutein, particularly as regardspotential secondary structures. Hosts should be selected byconsideration of their compatibility with the chosen vector, thetoxicity of the product coded for by the DNA sequences of thisinvention, their secretion characteristics, their ability to fold thepolypeptides correctly, their fermentation or culture requirements, andthe ease of purification of the products coded for by the DNA sequences.

Within these parameters one of skill in the art may select variousvector/expression control sequence/host combinations that will expressthe desired DNA sequences on fermentation or in large scale animalculture, for example, using CHO cells or COS 7 cells.

The choice of expression control sequence and expression vector, in someembodiments, will depend upon the choice of host. A wide variety ofexpression host/vector combinations can be employed. Useful expressionvectors for eukaryotic hosts, include, for example, vectors withexpression control sequences from SV40, bovine papilloma virus,adenovirus and cytomegalovirus. Useful expression vectors for bacterialhosts include known bacterial plasmids, such as plasmids from E. coli,including col El, pCRI, pER32z, pMB9 and their derivatives, wider hostrange plasmids, such as RP4, phage DNAs, e.g., the numerous derivativesof phage lambda, e.g., NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages. Useful expression vectors foryeast cells include the 2μ plasmid and derivatives thereof. Usefulvectors for insect cells include pVL 941 and pFastBac™ 1 (GibcoBRL,Gaithersburg, Md.). Cate et al., “Isolation Of The Bovine And HumanGenes For Mullerian Inhibiting Substance And Expression Of The HumanGene In Animal Cells”, Cell, 45, pp. 685-98 (1986).

In addition, any of a wide variety of expression control sequences canbe used in these vectors. Such useful expression control sequencesinclude the expression control sequences associated with structuralgenes of the foregoing expression vectors. Examples of useful expressioncontrol sequences include, for example, the early and late promoters ofSV40 or adenovirus, the lac system, the trp system, the TAC or TRCsystem, the major operator and promoter regions of phage lambda, forexample PL, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., PhoA, the promoters of the yeast a-matingsystem, the polyhedron promoter of Baculovirus, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells or their viruses, and various combinations thereof.

A T7 promoter can be used in bacteria, a polyhedrin promoter can be usedin insect cells, and a cytomegalovirus or metallothionein promoter canbe used in mammalian cells. Also, in the case of higher eukaryotes,tissue-specific and cell type-specific promoters are widely available.These promoters are so named for their ability to direct expression of anucleic acid molecule in a given tissue or cell type within the body.Skilled artisans are well aware of numerous promoters and otherregulatory elements which can be used to direct expression of nucleicacids.

In addition to sequences that facilitate transcription of the insertednucleic acid molecule, vectors can contain origins of replication, andother genes that encode a selectable marker. For example, theneomycin-resistance (neo^(r)) gene imparts G418 resistance to cells inwhich it is expressed, and thus permits phenotypic selection of thetransfected cells. Those of skill in the art can readily determinewhether a given regulatory element or selectable marker is suitable foruse in a particular experimental context.

Viral vectors that can be used in the invention include, for example,retroviral, adenoviral, and adeno-associated vectors, herpes virus,simian virus 40 (SV40), and bovine papilloma virus vectors (see, forexample, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press,Cold Spring Harbor, N.Y.).

Prokaryotic or eukaryotic cells that contain and express a nucleic acidmolecule that encodes a subject IL-2 mutein disclosed herein are alsofeatures of the invention. A cell of the invention is a transfectedcell, i.e., a cell into which a nucleic acid molecule, for example anucleic acid molecule encoding a mutant IL-2 polypeptide, has beenintroduced by means of recombinant DNA techniques. The progeny of such acell are also considered within the scope of the invention.

The precise components of the expression system are not critical. Forexample, an IL-2 mutein can be produced in a prokaryotic host, such asthe bacterium E. coli, or in a eukaryotic host, such as an insect cell(e.g., an Sf21 cell), or mammalian cells (e.g., COS cells, NIH 3T3cells, or HeLa cells). These cells are available from many sources,including the American Type Culture Collection (Manassas, Va.). Inselecting an expression system, it matters only that the components arecompatible with one another. Artisans or ordinary skill are able to makesuch a determination. Furthermore, if guidance is required in selectingan expression system, skilled artisans may consult Ausubel et al.(Current Protocols in Molecular Biology, John Wiley and Sons, New York,N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual,1985 Suppl. 1987).

The expressed polypeptides can be purified from the expression systemusing routine biochemical procedures, and can be used, e.g., astherapeutic agents, as described herein.

In some embodiments, IL-2 muteins obtained will be glycosylated orunglycosylated depending on the host organism used to produce themutein. If bacteria are chosen as the host then the IL-2 mutein producedwill be unglycosylated. Eukaryotic cells, on the other hand, willglycosylate the IL-2 muteins, although perhaps not in the same way asnative-IL-2 is glycosylated. The IL-2 mutein produced by the transformedhost can be purified according to any suitable method. Various methodsare known for purifying IL-2. See, e.g. Current Protocols in ProteinScience, Vol 2. Eds: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg,David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, JohnWiley and Sons, Inc. IL-2 muteins can be isolated from inclusion bodiesgenerated in E. coli, or from conditioned medium from either mammalianor yeast cultures producing a given mutein using cation exchange, gelfiltration, and or reverse phase liquid chromatography.

Another exemplary method of constructing a DNA sequence encoding theIL-2 muteins is by chemical synthesis. This includes direct synthesis ofa peptide by chemical means of the protein sequence encoding for an IL-2mutein exhibiting the properties described. This method can incorporateboth natural and unnatural amino acids at positions that affect theinteractions of IL-2 with the IL-2Rα, the IL-2Rβ and/or the IL-2Rγ.Alternatively a gene which encodes the desired IL-2 mutein can besynthesized by chemical means using an oligonucleotide synthesizer. Sucholigonucleotides are designed based on the amino acid sequence of thedesired IL-2 mutein, and preferably selecting those codons that arefavored in the host cell in which the recombinant mutein will beproduced. In this regard, it is well recognized that the genetic code isdegenerate—that an amino acid may be coded for by more than one codon.For example, Phe (F) is coded for by two codons, TIC or TTT, Tyr (Y) iscoded for by TAC or TAT and his (H) is coded for by CAC or CAT. Trp (W)is coded for by a single codon, TGG. Accordingly, it will be appreciatedthat for a given DNA sequence encoding a particular IL-2 mutein, therewill be many DNA degenerate sequences that will code for that IL-2mutein. For example, it will be appreciated that in addition to thepreferred DNA sequence for mutein 5-2 shown in FIG. 2 , there will bemany degenerate DNA sequences that code for the IL-2 mutein shown. Thesedegenerate DNA sequences are considered within the scope of thisdisclosure. Therefore, “degenerate variants thereof” in the context ofthis invention means all DNA sequences that code for and thereby enableexpression of a particular mutein.

The biological activity of the IL-2 muteins can be assayed by anysuitable method known in the art. Such assays include PHA-blastproliferation and NK cell proliferation.

Methods of Treatment

In some embodiments, subject IL-2 muteins, and/or nucleic acidsexpressing them, can be administered to a subject to treat a disorderassociated with abnormal apoptosis or a differentiative process (e.g.,cellular proliferative disorders or cellular differentiative disorders,such as cancer, by, for example, producing an active or passiveimmunity). In the treatment of such diseases, the disclosed IL-2 muteinsmay possess advantageous properties, such as reduced vascular leaksyndrome.

Examples of cellular proliferative and/or differentiative disordersinclude cancer (e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias). A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver. Thecompositions of the present invention (e.g., mutant IL-2 polypeptidesand/or the nucleic acid molecules that encode them) can also beadministered to a patient who has a viral infection (e.g., AIDS or aninfluenza).

The mutant IL-2 polypeptides can be used to treat patients who have, whoare suspected of having, or who may be at high risk for developing anytype of cancer, including renal carcinoma or melanoma, or any viraldisease. Exemplary carcinomas include those forming from tissue of thecervix, lung, prostate, breast, head and neck, colon and ovary. The termalso includes carcinosarcomas, which include malignant tumors composedof carcinomatous and sarcomatous tissues.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders.

Other examples of proliferative and/or differentiative disorders includeskin disorders. The skin disorder may involve the aberrant activity of acell or a group of cells or layers in the dermal, epidermal, orhypodermal layer, or an abnormality in the dermal-epidermal junction.For example, the skin disorder may involve aberrant activity ofkeratinocytes (e.g., hyperproliferative basal and immediately suprabasalkeratinocytes), melanocytes, Langerhans cells, Merkel cells, immunecell, and other cells found in one or more of the epidermal layers,e.g., the stratum basale (stratum germinativum), stratum spinosum,stratum granulosum, stratum lucidum or stratum corneum. In otherembodiments, the disorder may involve aberrant activity of a dermalcell, for example, a dermal endothelial, fibroblast, immune cell (e.g.,mast cell or macrophage) found in a dermal layer, for example, thepapillary layer or the reticular layer.

Examples of skin disorders include psoriasis, psoriatic arthritis,dermatitis (eczema), for example, exfoliative dermatitis or atopicdermatitis, pityriasis rubra pilaris, pityriasis rosacea, parapsoriasis,pityriasis lichenoiders, lichen planus, lichen nitidus, ichthyosiformdermatosis, keratodermas, dermatosis, alopecia areata, pyodermagangrenosum, vitiligo, pemphigoid (e.g., ocular cicatricial pemphigoidor bullous pemphigoid), urticaria, prokeratosis, rheumatoid arthritisthat involves hyperproliferation and inflammation of epithelial-relatedcells lining the joint capsule; dermatitises such as seborrheicdermatitis and solar dermatitis; keratoses such as seborrheic keratosis,senile keratosis, actinic keratosis, photo-induced keratosis, andkeratosis follicularis; acne vulgaris; keloids and prophylaxis againstkeloid formation; nevi; warts including verruca, condyloma or condylomaacuminatum, and human papilloma viral (HPV) infections such as venerealwarts; leukoplakia; lichen planus; and keratitis. The skin disorder canbe dermatitis, e.g., atopic dermatitis or allergic dermatitis, orpsoriasis.

Patients amenable to treatment may also have psoriasis. The term“psoriasis” is intended to have its medical meaning, namely, a diseasewhich afflicts primarily the skin and produces raised, thickened,scaling, nonscarring lesions. The lesions are usually sharply demarcatederythematous papules covered with overlapping shiny scales. The scalesare typically silvery or slightly opalescent. Involvement of the nailsfrequently occurs resulting in pitting, separation of the nail,thickening and discoloration. Psoriasis is sometimes associated witharthritis, and it may be crippling. Hyperproliferation of keratinocytesis a key feature of psoriatic epidermal hyperplasia along with epidermalinflammation and reduced differentiation of keratinocytes. Multiplemechanisms have been invoked to explain the keratinocytehyperproliferation that characterizes psoriasis. Disordered cellularimmunity has also been implicated in the pathogenesis of psoriasis.Examples of psoriatic disorders include chronic stationary psoriasis,psoriasis vulgaris, eruptive (gluttate) psoriasis, psoriaticerythroderma, generalized pustular psoriasis (Von Zumbusch), annularpustular psoriasis, and localized pustular psoriasis.

Alternatively, or in addition to methods of direct administration topatients, in some embodiments, mutant IL-2 polypeptides can be used inex vivo methods. For example, cells (e.g., peripheral blood lymphocytesor purified populations of lymhocytes isolated from a patient and placedor maintained in culture) can be cultured in vitro in culture medium andthe contacting step can be affected by adding the IL-2 mutant to theculture medium. The culture step can include further steps in which thecells are stimulated or treated with other agents, e.g., to stimulateproliferation, or to expand a population of cells that is reactive to anantigen of interest (e.g., a cancer antigen or a viral antigen). Thecells are then administered to the patient after they have been treated.

In certain embodiments, the subject IL-2 mutein that function as IL-2antagonists described herein are useful for the treatment of one or moreconditions wherein suppression of one or more IL-2 and/or IL-15dependent functions is useful. In certain embodiments, the IL-2 muteinantagonists described herein is used for the treatment of one or morediseases or conditions wherein suppression of IL-2Rβ/IL-2Rγheterodimerization and downstream signaling is useful (e.g., GVDH orleukemia).

In one embodiment, the method of treatment is for the treatment of graftversus host disease (GVHD). In some embodiments, the treatment includesthe step of administering to a subject having GVHD a therapeuticallyeffective amount of an IL-2 mutein that is an IL-2 antagonist. IL-2 andIL-15 is known to contribute to GVHD ((Ferrara et al., Journal ofImmunology 137: 1874 (1986); and Blaser et al., Blood 105: 894 (2005)).Therefore, without being bound by any particular theory of operation, itis believed that the IL-2 antagonists described herein are useful forthe treatment of GVHD. In one embodiment, the IL-2 mutein for thetreatment of GVHD includes one or more mutations that reduces itsbinding to IL-2Rγ_(c) receptor as compared to wild type IL-2 (e.g., anyone of the mutations that reduces IL-2Rγ_(c) receptor binding asdescribed herein). In some embodiments, the mutations that decreaseIL-2Rγ_(c) receptor binding affinity include the amino acidsubstitutions L18R, Q22E, Q126T and S130R. In some embodiments, the IL-2mutein further with decreased IL-2Rγ_(c) receptor binding affinityfurther includes one or more amino acid mutations that increase the IL-2muteins binding affinity for IL-2Rβ, as compared to wild-type IL-2(e.g., any one of the mutations that increase IL-2Rβ binding asdescribed herein). In some embodiments, the IL-2 mutein further includesthe amino acid substitutions L80F, R81D, L85V, I86V and I92F. In someembodiments, the IL-2 mutein includes the amino acid substitutions L18R,Q22E, L80F, R81D, L85V, Q126T, S130R, 186V and 192F.

In another embodiment, the method of treatment is for the treatment ofan IL-2 and/or IL-15 mediated leukemia. In particular embodiments, theleukemia is adult T-cell leukemia (ATL). ATL is characterized by amalignant expansion of CD4+ T cells that exhibit an early growth phasethat involves autocrine signals by IL-2 and IL-15 as well as paracrinesignals by IL-9. Such cytokine-dependent proliferation is evident inpatients with chronic and smoldering but not acute ATL. Therefore,without being bound by any particular theory of operation, it isbelieved that IL-2 partial agonists and antagonists can be used to treatsuch forms of leukemia. In some embodiments, the treatment includes thestep of administering to a subject having adult T-cell leukemia atherapeutically effective amount of an IL-2 mutein that is an IL-2antagonist. In some embodiments, the patient has chronic and smolderingATL. In one embodiment, the IL-2 mutein for the treatment of ATLincludes one or more mutations that reduces its binding to IL-2R γ_(c)receptor as compared to wild type IL-2 (e.g., any one of the mutationsthat reduces IL-2R γ_(c) receptor binding as described herein). In someembodiments, the mutations that decrease IL-2R γ_(c) receptor bindingaffinity include the amino acid substitutions L18R, Q22E, Q126T andS130R. In some embodiments, the IL-2 mutein further with decreasedIL-2Rγ_(c) receptor binding affinity further includes one or more aminoacid mutations that increase the IL-2 muteins binding affinity forIL-2Rβ, as compared to wild-type IL-2 (e.g., any one of the mutationsthat increase IL-2Rβ binding as described herein). In some embodiments,the IL-2 mutein further includes the amino acid substitutions L80F,R81D, L85V, I86V and I92F. In some embodiments, the IL-2 mutein includesthe amino acid substitutions L18R, Q22E, L80F, R81D, L85V, Q126T, S130R,186V and 192F.

Pharmaceutical Compositions and Methods of Administration

In some embodiments, subject IL-2 muteins and nucleic acids can beincorporated into compositions, including pharmaceutical compositions.Such compositions typically include the polypeptide or nucleic acidmolecule and a pharmaceutically acceptable carrier.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. The mutant IL-2 polypeptides of theinvention may be given orally, but it is more likely that they will beadministered through a parenteral route. Examples of parenteral routesof administration include, for example, intravenous, intradermal,subcutaneous, transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral applicationcan include the following components: a sterile diluent such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. pH can be adjusted with acids or bases, such asmono- and/or di-basic sodium phosphate, hydrochloric acid or sodiumhydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5). The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. It should be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants,e.g., sodium dodecyl sulfate. Prevention of the action of microorganismscan be achieved by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol,sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions, if used, generally include an inert diluent or anedible carrier. For the purpose of oral therapeutic administration, theactive compound can be incorporated with excipients and used in the formof tablets, troches, or capsules, e.g., gelatin capsules. Oralcompositions can also be prepared using a fluid carrier for use as amouthwash. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel™, or corn starch; a lubricant such as magnesium stearate orSterotesrm; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In the event of administration by inhalation, subject IL-2 muteins, orthe nucleic acids encoding them, are delivered in the form of an aerosolspray from pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer. Suchmethods include those described in U.S. Pat. No. 6,468,798.

Systemic administration of the subject IL-2 muteins or nucleic acids canalso be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

In some embodiments, compounds (mutant IL-2 polypeptides or nucleicacids) can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In some embodiments, compounds (subject IL-2 muteinsor nucleic acids)can also be administered by transfection or infection using methodsknown in the art, including but not limited to the methods described inMcCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol.20: 1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151-160,1996, erratum at Am. J. Health Syst. Pharm. 53:325, 1996).

In one embodiment, the subject IL-2 muteins or nucleic acids areprepared with carriers that will protect the mutant IL-2 polypeptidesagainst rapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Such formulations can be preparedusing standard techniques. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Dosage, toxicity and therapeutic efficacy of such subject IL-2 muteinsor nucleic acids compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds thatexhibit high therapeutic indices are preferred. While compounds thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such compounds to the site of affectedtissue in order to minimize potential damage to uninfected cells and,thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of a subject IL-2mutein (i.e., an effective dosage) depends on the polypeptide selected.For instance, single dose amounts in the range of approximately 0.001 to0.1 mg/kg of patient body weight can be administered; in someembodiments, about 0.005, 0.01, 0.05 mg/kg may be administered. In someembodiments, 600,000 IU/kg is administered (IU can be determined by alymphocyte proliferation bioassay and is expressed in InternationalUnits (IU) as established by the World Health Organization 1^(st)International Standard for Interleukin-2 (human)). The dosage may besimilar to, but is expected to be less than, that prescribed forPROLEUKIN®. The compositions can be administered one from one or moretimes per day to one or more times per week; including once every otherday. The skilled artisan will appreciate that certain factors mayinfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of the subject IL-2 muteins can includea single treatment or, can include a series of treatments. In oneembodiment, the compositions are administered every 8 hours for fivedays, followed by a rest period of 2 to 14 days, e.g., 9 days, followedby an additional five days of administration every 8 hours.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The following examples are provided to describe certain embodiments ofthe invention provided herein and are not to be construed to aslimiting.

EXAMPLES Example 1: Functional Expression of IL-2 on the Surface ofYeast

Although IL-2 has been displayed on bacteriophage previously (Buchli etal., Arch. Biochem. Biophys. 339:79-84, 1997), the prior system was notamenable to directed evolution and therefore not suitable for obtainingIL-2 mutants with improved binding for subunits of the IL-2R. Toovercome this, IL-2 was expressed on the surface of yeast cells. HumanIL-2 DNA was cloned into yeast display vector pCT302. Saccharomycescerevisiae strain EBY100 was transformed with the pCT302_IL-2 vector andgrown for 3 days at 30° C. on SD-CAA plates. Individual colonies of IL-2yeast were grown overnight at 30° C. in SD-CAA, then introduced in SGCAAfor 2 days at 20° C. The yeast were stained with tetramerizedbiotinylated IL-2Rβ, biotinylated γ or biotinylated IL-2Rβ in thepresence of biotinylated γ. The ectodomains of IL-2Rβ and γ wereC-terminally biotinylated and coupled to phycoerythrin-conjugatedstrepavidin for use as a staining and sorting reagent. IL-2 Rβ tetramerswere formed by incubating 2 μM of biotinylated IL-2Rβ with 470 nMstreptavidin-phycoerythrin (SA-PE, Invitrogen) for 15 minutes on ice.These receptor “tetramers” enhanced the avidity of the low affinitymonomeric ectodomain (ECD) interactions with IL-2, enabling maximalrecovery of IL-2 variants from libraries. Similar to solution wild-typeIL-2, yeast-displayed IL-2 bound weakly to IL-2Rβ alone, did not bind toat all to γ alone, but did bind to γ in the presence of IL-2Rβ, asevidenced by diagonal staining seen by flow cytometry (data not shown).Thus, the yeast-displayed IL-2 recapitulates the cooperative assembly ofthe heterodimeric receptor complex on cells seen with soluble IL-2, andis therefore suitable as a platform for library selection.

Example 2: Construction and Screening of an IL-2 Mutant Library

The first generation in vitro strategy was to create an error-prone PCRlibrary of the entire IL-2 gene. The first generation mutant IL-2library was constructed as follows. Wildtype human interleukin-2 (IL-2)was subjected to error-prone mutagenesis using the GeneMorph® II RandomMutagenesis kit following the manufacturer's instructions. The followingprimers were used for error-prone PCR: 5′-GCACCTACTTCAAGTTCTAC-3′(“IL-2_errprone_for) and 5′-GCCACCAGAGGATCC-3′ (“IL-2_errprone_rev). Theproduct of the error prone PCR reaction was then amplified using thefollowing primers:5′AGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCGCACCTACTTCAAGTTCTAC-3′ (SEQ ID NO: 33) and5′ACACTGTTGTTATCAGATCTCGAGCAAGTCTTCTTCGGAGATAAGCTITTGTTCGCCACCAGAGGATCC-3′ (SEQ ID NO: 34) to yield approximately 130 μg of DNA.Yeast display vector pCT302 was double digested with restriction enzymesNheI and BamHI and gel purified. The IL-2 DNA and the pCT302 DNA weremixed together in a 5:1 μg ratio with electrocompetent EBY100 yeast. Theyeast were electroporated to facilitate entry of the library DNA intothe yeast. This electroporation was repeated approximately 20 times toyield a final library size of 1×10S transformants.

Selection of first generation IL-2 library: The library was subjected tosix rounds of selection against IL-2Rβ (FIG. 2A). In the first round,the library was labeled with 470 nM tetrameric IL-2Rβ, which was formedby mixing 2 μM biotinylated IL-2Rβ with 470 nMstreptavidin-phycoerythrin conjugate (SAV-PE) for 15 min. The librarywas incubated with IL-2Rβ for 1.5 h, washed with PBS-BSA buffer(phosphate buffered saline+bovine serum albumin), and incubated withMiltenyi anti-PE MicroBeads for 20 min at 4° C. The cells were againwashed and flowed over a magnetic column for selection. This selectionmethod was successively repeated five more times with alterations onlyin IL-2Rβ concentration (round 2-1 μM, round 3-1 μM, round 4-300 nM,round 5-300 nM, round 6-100 nM, all monomeric IL-2Rβ). Upon conclusionof selections, round five and round six yeast cultures were spread onSD-CAA plates, which yielded individual yeast colonies. Eighteenresulting yeast colonies were tested for binding to 500 nM IL-2Rβ. TheIL-2 DNA isolated from these eighteen yeast colonies was sequenced.Amino acid differences among these eighteen yeast colonies relative tothe corresponding residue in wildtype IL-2 is shown in Table 1.

TABLE 1 residue # 5 34 43 61 74 75 77 81 85 103 106 112 120 wt IL-2 S PK E Q S N R L F E A R 5_1 R 5_2 V 5_3 V D 5_4 K Y 5_5 V 5_6 R S 5_8 (wt)5_9 R V 5_10 V 6_1 V 6_2 R R 6_3 N V 6_4 V 6_5 V 6_6 I V 6_7 I V 6_8 K V6_10 T VLibrary construction of second generation IL-2 library: Based on thehigh percentage of clones containing L85V, a second IL-2 library wasconstructed that focused primarily on hydrophobic core residues. Asite-directed IL-2 library was constructed with mutations at Q74, L80,R81, L85, I86, I89, I92, V93. Q74 was allowed to vary as H/K/N/Q/R/S.R81 was allowed to vary at all 20 amino acids with the NNK degeneratecodon, where N represents a 25% mix each of adenine, thymine, guanine,and cytosine nucleotides and K is either guanine or thymine. Theremaining residues were allowed to vary as F/I/UV. The library wasconstructed by assembly PCR using the following oligos:

IL-2_affmat_ass01 (SEQ ID NO: 35)GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCA IL-2_affmat_ass02(SEQ ID NO: 36) CAAAATCATCTGTAAATCCAGAAGTAAATGCTCCAGTTGTAGCTGTGIL-2_affmat_ass03 (SEQ ID NO: 37)GGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCA IL-2 affmat_ass04B(SEQ ID NO: 38) AACTTAGCTGTGAGCATCCTGGTGAGTTTGGGATTCTTGTAATTATTIL-2_affmat_ass05B (SEQ ID NO: 39)GGATGCTCACAGCTAAGTTTTACATGCCCAAGAAGGCCACAGAACTG IL-2_affmat_ass06(SEQ ID NO: 40) GTTCTTCTTCTAGACACTGAAGATGTTTCAGTTCTGTGGCCTTCTTGIL-2_affmat_ass07 (SEQ ID NO: 41CAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTA IL-2 affmat_ass08(SEQ ID NO: 42) GTGAAAGTTTTTGCT SYK AGCTAAATTTAGCACTTCCTCCIL-2_affmat_ass09 (SEQ ID NO: 43) AGCAAAAACTTTCAC NTCNNK CCCAGGGACNTCNTC AGCAAT NTC AACGTA NTCNTC CTGGAACTAAAGGGATC IL-2_affmat_ass10(SEQ ID NO: 44) CATCAGCATATTCACACATGAATGTTGTTTCAGATCCCTTTAGTTCCAGIL-2_affmat_ass11 (SEQ ID NO: 45)ATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACA IL-2_affmat_ass12(SEQ ID NO: 46) AGATGATGCTTTGACAAAAGGTAATCCATCTGTTCAGAAATTCTACAATIL-2_affmat_ass13 (SEQ ID NO: 47)TTTTGTCAAAGCATCATCTCAACACTAACTGGATCCTCTGGTGGCThe site-directed PCR was amplified with the following oligos:

PCR Amplification Oligos (Including 50 bp Homology)

IL-2_site2_assFor: (SEQ ID NO: 48) 5′-AGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCGCACCTACTTCAAGTTCTAC-3′ IL-2_site2_assRev: (SEQ ID NO: 49) 5′-ACACTGTTGTTATCAGATCTCGAGCAAGTCTTCTTCGGAGATAAGCTTTTGTTCGCCACCAGAGGATCC-3′The PCR yielded 40 μg of DNA, which was mixed with double digestedpCT302 and electrocompetent EBY100 yeast and electroporated as with thefirst generation library.Selection of second generation IL-2 library: The library was subjectedto five rounds of selection against IL-2Rβ (FIG. 2B). This selectionmethod was performed exactly as with the first generation library, onlywith modifications to the concentrations of IL-2Rβ used (round 1-1 μM,round 2-100 nM, round 3-30 nM, round 4-30 nM, round 5-10 nM, allmonomeric IL-2Rβ). Upon conclusion of selections, round four and roundfive yeast cultures were spread on SD-CAA plates, which yieldedindividual yeast colonies. 48 individual yeast clones from both roundswere grown in 96-well block format and screened by labeling with 5 nMIL-2Rβ and then SAV-PE. The screen yielded seven high affinity bindersto IL-2Rβ (FIG. 3 and Table 2). Amino acid differences among these sevenhigh affinity binders relative to the corresponding residue in wildtypeIL-2 is shown in Table 2 along with the binding affinity for IL-2Rβ.

TABLE 2 residue # 74 80 81 85 86 89 92 93 K_(d) (nM) wt IL-2 Q L R L I II V 280 B1 N F D V V V F 1.6 C5 N V T V V F 10 D10 H F D V V F 1.2 E10 SF D V V F 1.3 G8 N F D V V F 1.5 H4 S T V F 14 H9 F D V V F 1.3CONSENSUS F D V V F

Example 3: IL-2 Mutein Protein Expression and Purification

Human IL-2 variants (amino acids 1-133), the IL-2Rβ ectodomain (aminoacids 1-214), and γ_(c) (amino acids 34-232) were cloned into thepAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67signal sequence and C-terminal hexahistidine tag and produced using thebaculovirus expression system. Baculovirus stocks were prepared bytransfection and amplification in Spodoptera frugiperda (Sf9) cellsgrown in SF900II media (Invitrogen), and protein expression was carriedout in suspension Trichoplusia ni (High Five™) cells grown inBioWhittaker® Insect XPRESS™ media (Lonza). Proteins were expressed andcaptured from High Five™ supernatants after 48-60 hr by nickel agarose(QIAGEN), concentrated and purified by size exclusion chromatography ona Superdex™ 200 column (GE Healthcare), equilibrated in 10 mM HEPES (pH7.2) and 150 mM NaCl. IL-2 variants used in SPR and cell based assayswere expressed fully glycoslylated. For biotinylated receptorexpression, IL-2Rβ and γ_(c) were cloned into the pAcGP67-A vector witha C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE andhexahistidine tag. Receptor proteins were coexpressed with BirA ligasewith excess biotin (100 uM).

Example 4: Stimulation of CD25⁻ and CD25⁺ Natural Killer (YT-1) Cells

YT-1 and CD25+YT-1 cells were cultured in RPMI 1640 medium supplementedwith 10% fetal bovine serum, 2 mM L-glutamine, minimum non-essentialamino acids, sodium pyruvate, 25 mM HEPES, and penicillin-streptomycin(Gibco). CD25⁺ YT-1 cells were purified as follows: 1×10⁷ cells werewashed with FACS buffer (phosphate buffered saline+2% bovine serumalbumin) and stained with PE-conjugated anti-human CD25 (1:20;Biolegend, San Diego, Calif.) in 1 mL FACS buffer for 20 minutes at 4°C. The stained cells were labeled with paramagnetic microbeads coupledto anti-PE IgG and separated with an LS MACS® separation columnaccording to the manufacturer's instructions (Miltenyi Biotec, BergischGladbach, Germany). Eluted cells were re-suspended in complete RPMImedium at a concentration of 1×10⁵ cells and expanded for subsequentexperiments. Enrichment of cells was monitored via flow cytometry withthe FL-2 channel using an Accuri® C6 flow cytometer.

The dose-response relationships of H9, D10, and 6-6 on YT-1 cells wasdetermined by assaying STAT5 phosphorylation with flow cytometry (FIGS.4A and 4B). CD25⁺ or CD25⁻ YT-1 cells were washed with FACS buffer andre-suspended in 200 μL FACS buffer with the indicated concentration ofwild-type, 6-6, H9, or D10 in a 96 well plate. Cells were stimulated for20 minutes at room temperature and then fixed by addition offormaldehyde to 1.5% and incubated for 10 min. Cells were permeabilizedwith 100% ice-cold methanol for 20 min on ice, followed by incubation at−80° C. overnight. Fixed, permeabilized cells were washed with excessFACS buffer and incubated with 50 μL Alexa647 conjugated anti-STAT5pY694 (BD Biosciences, San Jose, Calif.) diluted 1:20 in FACS buffer for20 minutes. Cells were washed twice in FACS buffer and mean cellfluorescence determined using the FL-4 channel of an Accuri® C6 flowcytometer.

The CD25 independence of the IL-2 muteins (so called “super-2”molecules) was further tested by taking advantage of awell-characterized mutation of IL-2, phenylalanine to alanine atposition 42 (F42A), which abolishes binding to CD25, yet does not effectits ability to bind to the IL-2Rβ or the IL-2Rγ (Mott, 1995). Thismutation was also introduced into the H9 mutein, yielding H9 F42A. Acomparison of STAT induction by IL-2, IL-2 F42A, H9 and H9 F42A on CD25⁻and CD25+YT-1 cells was performed (FIG. 5 ). While IL-2 F42A mutationright shifted the dose response curve of wild-type IL-2 on CD25+ cellsby approximately 1 log, the F42A mutation had no observable effect onSTAT induction on CD25− cells (FIG. 5A). In contrast, the dose responsecurves of H9 and H9 F42A were essentially overlapping on both CD25⁻ andCD25+ cells (FIG. 5B). Thus, these experiments demonstrate that whilethe IL-2 muteins do not apparently benefit from the presence of CD25,their activity is insensitive to mutations that disrupt the CD25interface.

Example 5: Stimulation of CD25⁻ and CD25+ T Cells

Human and mouse CD4 T cells were prepared from PBMC (Stanford BloodBank) and spleens and lymph nodes of BALB/C mice, respectively usingantibody-coated CD4 T cell isolation magnetic beads (Stem CellTechnologies and Miltenyi Biotec). For naïve cell stimulation assays,cells were used immediately. For generation of in vitro ‘experienced’ Tcells, wells were pre-coated with secondary antibody (Vector Labs) inbicarbonate buffer, pH 9.6 prior to coating plates with anti-CD3 (OKT3for human, 2C11 for mouse, eBiosciences) at 100 ng/mL. T cells wereseeded at 0.1×10⁶ cells/well with soluble anti-CD28 (CD28.2 for human,37.51 for mouse, eBiosciences). Cell were cultured for 3 days with fullTCR stimulated, followed by 2 days rest in conditioned media and 2 daysrest in fresh culture media. Prior to use, live cells were collected byLympholyte-M (Cederlane) centrifugation and counted.

The activity of IL-2 muteins on T cells that were either deficient inCD25 expression or expressed CD25 was assessed (FIG. 6 ). The doseresponse relationship of wild-type IL-2 and six IL-2 muteins wereassayed for STAT5 phosphorylation at a protein concentration range of 1ng/ml to 1000 ng/ml. The ability of the IL-2 muteins to stimulate STAT5phosphorylation in CD25 deficient T cells correlated well with theiraffinity for the IL-2Rβ. The increase in STAT5 phosphorylation by theIL-2 muteins was two orders of magnitude greater that IL-2.

The ability of IL-2 muteins to stimulate STAT5 phosphorylation onexperienced human CD4+ T cells, which express large amounts of the fullIL-2 receptor complex, CD25 (IL-2Rβ), IL-2Rβ, and γ_(c) was alsoassessed (FIG. 7 ). Human CD4 T cells were in vitro TCR stimulated andrested to generate ‘experienced’ human CD4+CD25+ T lymphocytes. At 1ng/mL, almost no difference in STAT5 phosphorylation was observed. EachIL-2 variant, including wild-type, stimulated over 90% of the cells. At0.1 ng/mL, small differences were observed. Wildtype IL-2 resulted in48% pSTAT5 stimulation, and the IL-2 muteins yielded between 65-79%pSTAT5 stimulation. Therefore, the IL-2 muteins apparently stimulateexperienced human T cells better than wildtype IL-2 but the enhancementis not as pronounced as on cells lacking CD25

Example 6: NK Cell Cytotoxicity Assay

The effect of the D10 IL-2 mutein on Natural Killer cell function,specifically spontaneous and antibody-dependent cell-mediatedcytotoxicity (ADCC) using an EGFR (endothelial growth factorreceptor)-expressing squamous tumor cell line (SCC6) and the EGFRmonoclonal antibody, cetuximab was assessed. Human EGFR-positivesquamous cell carcinoma cell line, SCC6, was obtained as a gift from theJ. Sunwoo Laboratory (Stanford, Calif.). SCC6 cell line was cultured inDMEM/F12 medium (Invitrogen Life Technologies) supplemented with 10%heat-inactivated FCS (HyClone Laboratories), 100 U/mL penicillin and 100μg/mL streptomycin (both from Invitrogen Life Technologies). Cells weregrown adherent in culture at 37° C. in 5% CO2. Cetuximab (mouse chimericIgG1 anti-human epidermal growth factor receptor-EGFR; IMC-C225;Erbitux®) was obtained from Bristol-Myers Squibb.

Chromium release was performed as follows: NK cells were isolated from ahealthy donor leukocyte-reduced system (LRS) product containingapproximately 1×10⁹ cells. NK cells were isolated by negative magneticcell sorting using NK cell isolation beads (Miltenyi Biotec) accordingto manufacturer's instructions. NK cells were assessed for purity (>90%purity as defined by CD3⁻CD56⁺ flow cytometry). SCC6 target cells werelabeled with 150 μCi ⁵¹Cr per 1×10⁶ cells for 2 h. Percent lysis wasdetermined after 5 h cultures of purified NK cells at variableeffector:target cell ratios of 0:1, 1:1, and 5:1 with ⁵¹Cr-labeled SCC6cells in media alone, cetuximab (100 μg/mL), IL-2 (1000 IU/mL), IL-2 D10(1 μg/mL), IL-2 D10 (10 μg/mL), or combinations including cetuximab (100μg/mL) plus IL-2(1000 IU/mL), cetuximab (100 pg/mL) plus IL-2 D10 (1μg/mL), or cetuximab (100 μg/mL) plus IL-2 D10 (10 μg/mL). Assay wasperformed in triplicate. Purified NK cells were cultured with ⁵¹Crlabelled-SCC6 cells in the presence or absence of cetuximab, IL-2 orIL-2 D10 at variable concentrations. D10 stimulation of NK cellspontaneous cytotoxicity was superior to high-dose IL-2 (FIG. 8 ,*p=0.008, **p=0.001) with minimal spontaneous cytotoxicity without IL-2or D10 stimulation. ADCC of cetuximab-bound SCC6 was similarly increasedby D10 stimulation compared to high-dose IL-2 or cetuximab alone(*p=0.0005, **p=0.0001). Notably, superior functional enhancement ofcytotoxicity, both spontaneous and ADCC, occurred at all effector:targetratios including 1:1 with D10 compared to high-dose IL-2.

Example 7: IL-2 Muteins Result in Enhanced Memory Phenotype Expansionwith Relatively Low Stimulation of Suppressor-Type T Cells (Tregs)

The potency of the IL-2 mutein H9 on the expansion of memory phenotypeCD8⁺ T cells expressing low levels of CD25 but high levels of IL-2Rβγwas assessed in vivo. C57Bl/6 mice received either PBS, 20 μg IL-2, 20μg H9, or 1.5 μg IL-2/anti-IL-2 monoclonal antibody complexes and totalcell counts of splenic CD3⁺ CD4⁺ CD44^(high) memory phenotype T cellswere assessed by flow cytometry. Splenic cell suspensions were preparedand stained with fluorochrome-conjugated monoclonal antibodies CD3(clone 145-2C11, eBioscience), CD4 (clone RM4-5, Caltag Laboratories),CD8a (clone 53-6.7, BD Biosciences), CD25 (clone PC61, BD Biosciences),CD44 (clone IM7, eBioscience) NK1.1 (clone PK136, BD Biosciences) andThy1.1 (clone HIS51, eBioscience). At least 100,000 viable cells wereacquired using a BD FACSCanto™ II flow cytometer and analyzed usingFlowJo software (TriStar, Inc.). As shown in FIG. 10A, treatment withthe disclosed IL-2 mutein resulted in greater expansion of memoryphenotype T cells relative to other treatment modalities with limitedexpansion of CD3⁺ CD4⁺ CD25^(high) T cells regulatory T cells (FIG.10B).

Example 8: Reduced In Vivo Toxicity of IL-2 Muteins

It is known that IL-2 treatment can lead to severe adverse effects, suchas acute pulmonary edema, which is currently a limitation preventingmore effective use of IL-2. Accordingly, the toxicity of the disclosedIL-2 muteins relative to IL-2 was assessed (FIG. 11A). C57Bl/6 micereceived daily intraperitoneal injections of PBS, 20 μg IL-2, 20 μg H9,or 1.5 μg IL-2/anti-IL-2 monoclonal antibody complexes for 5 consecutivedays. 6 days after adoptive cell transfer, the lungs were removed andweighed before and after drying overnight at 58° C. under vacuum.Pulmonary wet weight was calculated by subtracting initial pulmonaryweight from lung weight after dehydration.

Example 9: Increased Anti-Tumor Activity of IL-2 Muteins In Vivo

The potency of the disclosed IL-2 muteins against tumor cells was testedin vivo. 10⁶ B16F10 melanoma cells in 100 μl RPMI were injected into theupper dermis in the back of mice (3-4 mice per group). Treatmentconsisted of five daily injections of either PBS, 20 μg IL-2, 20 pg H9,or 1.5 μg IL-2/anti-IL-2 monoclonal antibody complexes (IL-2/mAb) andwas started one day after tumor nodules were clearly visible andpalpable at a size of ˜15 mm². The disclosed IL-2 mutein resulted inenhanced anti-tumor activity in vivo as demonstrated in FIG. 11B.

Example 10: Structural Comparison of IL-2 Muteins and IL-2

Several of the IL-2 muteins were recombinantly expressed in order tomeasure their binding affinity and kinetics for IL-2Rβ by surfaceplasmon resonance (SPR). The affinity between IL-2 and IL-2Rβ wasK_(D)=280 nM. The IL-2 muteins clustered into low, medium, and highaffinity classes. The low affinity IL-2 muteins (5-2 and 6-6) boundIL-2Rβ with K_(D) between 50 and 70 nM, respectively, an affinity gainof 4-6 fold from wild-type IL-2 almost entirely through the L85Vsubstitution. The medium and high affinity mutants selected from thesecondary, site-directed library had K_(D)'S of 10-15 nM (C5, H4), and1.2-1.7 nM (B1, D10, E10, G8, H9), respectively. The affinity increaseswere uniformly manifested in reductions in off-rate, and the highaffinity IL-2 muteins contained a consensus sequence in the randomizedpositions of L80F/R81D/L85V/I86V/I92F.

To understand the structural consequences of the IL-2 muteins, the D10mutein as well as the ternary complex of D10 bound to IL-2Rβ and γ_(c)were crystallized. In the structure of D10 alone, five of the sixmutations are clustered on the B-C loop and within the C helix core, inpositions that do not contact IL-2Rβ. Notably, the B-C helix linkerregion is well-ordered in the electron density map (FIG. 9 ), comparedto other IL-2 structures where this region is either partially orcompletely disordered. Collectively, the F80, V85, and V86 substitutionsappear to collapse into a hydrophobic cluster that stabilizes the loopand ‘pins’ the C-helix into the core of the molecule, packing againsthelix B. The H74 and D81 mutations are solvent exposed and thus, theirstructural roles are less clear, however Asp is a well-known helixN-capping residue that could further contribute the helix C structure.Only one of the six consensus mutations, I92F, was at a position thatcontacts IL-2Rβ in the receptor complex. Phe92 is deeply insertedbetween the C and A helices, contributing only an additional 10 Å² ofmolecular surface buried by IL-2Rβ in the complex compared to I1e92.Thus, its IL-2Rβ contact likely makes only a small contribution to theoverall ˜300-fold affinity gain of D10.

A low-resolution (3.8 Å) structure of the D10 ternary receptor complexwas also determined to assess whether the mutations have perturbed theIL-2Rβ/γ_(c) receptor docking geometry. A stable ternary complex of D10and IL-2Rβ, was crystallized and purified in the absence of CD25. Theoverall IL-2Rβ/γ_(c) heterodimeric architecture and mode ofcytokine/IL-2Rβ contact in the D10 ternary complex is essentiallyidentical to the previously reported quaternary assembly. Thus, thepotency increase of super-2 is not due to a structural change inreceptor dimer architecture, but is likely due to the affinityenhancement.

As discussed earlier, the C-helix of IL-2 appears to undergo subtlerepositioning upon binding to IL-2Rα, as seen in both the binary andquaternary complexes. In contrast, inspection of three wild-typeunliganded structures in the PDB database reveals variability in theC-helix position, consistent with higher B-factors in this helixrelative to the rest of the molecule. Comparison of the structure of D10to that of an unliganded IL-2, and IL-2 in the receptor complexes wasundertaken. It was observed that the C-helix in D10 is more similar tothat seen in the two receptor-bound conformations of IL-2 than the freeforms, having undergone a shift up and into the helical core.

Molecular dynamics (MD) simulations were used to interrogate themechanism by which an IL-2 mutein is endowed with higher bindingaffinity for IL-2Rβ. An atomically detailed Markov state model (MSM) wasconstructed in order to directly probe the relative conformationalflexibility of IL-2 versus IL-2 muteins. The states in this MSM comefrom kinetic clustering of rapidly inter-converting conformationsresulting from atomistic simulations. Each of these metastable statescorresponds to a local minimum in the underlying free energy landscapethat ultimately determines the systems' structure and dynamics. Analysisof the MSM demonstrates that an IL-2 muetin can be more stable thanIL-2, and that IL-2 visits nearly twice as many clusters as an IL-2mutein. For example, IL-2 muteins most populated state has anequilibrium probability of ˜0.20, compared to ˜0.05 for IL-2. Helix B,the B-C loop, and helix C are rigidified in the IL-2 mutein compared toIL-2. As the evolved mutations reside on the B-C loop (H74, D81), andwithin the B and C helix packing interface (F80, V85, V86), bothhelices—not just helix C—benefit from the mutations and undergo acollective stabilization. F92 appears to act as a molecular wedgebetween helix C and helix A, acting as an additional stabilizinginfluence at the more C-terminal end of the helix. That the MDsimulations implicate helix B as also undergoing stabilization insuper-2 was a surprise, since this was not evident from comparison ofIL-2 crystal structures. IL-2Rα binds to IL-2 primarily on the B helixand part of the D helix. The MD simulations suggest the possibility thatbinding of IL-2Rα to IL-2 may rigidify helix B, and this structuralstabilization may be propagated to the B-C loop and helix C. Similar, inprinciple, to the apparent effect of the evolved mutations in the IL-2mutein.

Visualization of the most highly populated conformations from thesimulations for each protein shows that helix C, is far more flexible inIL-2 than the IL-2 mutein, and also that the mutations in the IL-2mutein do indeed stabilize a receptor-bound-like conformation.

Example 11: Partial IL-2 Agonists and Antagonists

IL-2 “superkines” with augmented action due to enhanced binding affinityfor IL-2Rβ were previously developed (Levin et al., Nature 484: 529(2012)). It was hypothesized that this high-affinity superkine/IL-2Rβcomplex could serve as a dominant-negative scaffold to create a“receptor signaling clamp” to block endogenous signaling. Directedmutation of these super-IL-2 “full agonists” to diminish binding toγ_(c) would attenuate IL-2Rβ-γ_(c) heterodimerization and represent anew class of mechanism-based IL-2 partial agonists and non-signaling(neutral) molecules that functionally act as antagonists by blockingendogenous cytokines and exerting no action of their own (see schematicin FIG. 1 ).

Based on the IL-2-IL-2R crystal structure, four key residues at theIL-2-γ_(c) interface were identified (FIG. 12A) and H9 variants weregenerated, each H9 variant containing one (Q126T), two (L18R, Q22E),three (L18R, Q22E, and Q126T), or four (L18R, Q22E, Q126T, and S130R)mutations (denoted as H9-T, H9-RE, H9-RET, and H9-RETR, respectively,based on the newly introduced amino acids). By surface plasmonresonance, recombinant H9 and H9-RET proteins had similar affinities forIL-2Rβ. However, whereas the H9-IL-2Rβ complex efficiently bound γ_(c)(FIG. 12B), H9-RET-IL-2Rβ did not (FIG. 12C).

To determine the activity of the H9 variants, CD25⁺ and CD25⁻subpopulations of NK-like YT-1 cells were purified and signaling wasquantified. The H9 mutants behaved as IL-2 partial agonists, producing arange of signaling efficacies from ˜90% down to ˜10% of the wild-typeE_(max) (maximum possible effect) level of phosphorylation of STAT5(FIG. 13A), with the level of activity for each analogue inverselycorrelating with the degree of mutation at the γ_(c) interface. Thesignaling potency was relatively independent of IL-2Rα, as demonstratedby the relative E_(max) values for H9-RET and H9-RETR on CD25⁺ (FIG.13B) versus CD25⁻ (FIG. 13C) YT-1 cells, suggesting that altered bindingto IL-2Rβ and γ_(c) was primarily responsible for the behavior of the H9variants.

H9-RET and H9-RETR also exhibited diminished induction of other IL-2signaling pathways, including pERK1/ERK2 in CD25⁺ YT-1 cells (FIG. 14A).As receptor internalization is a key event in signaling, surfaceexpression of IL-2Rβ and IL-2Rγ in YT-1 cells in response to the IL-2variants were also evaluated. Like IL-2 and H9, both H9-RET and H9-RETRdrove rapid and complete IL-2Rβ internalization (FIG. 13B), but thesepartial agonists were much less efficient at promoting internalizationof γ_(c) (FIG. 13C), consistent with their diminished binding to γ_(c).Thus, variably disrupting the γ_(c)-binding interface of H9 can yield avariety of IL-2 partial agonists, potentially with an extensiverepertoire of signaling efficacies.

Next, the effects of these molecules in primary cells were analyzed. Itwas determined that neither H9-RET nor H9-RETR could induce pSTAT5 infreshly isolated CD8⁺ T cells (FIGS. 13 , E and F, top panels), whichexpress less IL-20 and IL-2Rγ than activated CD8⁺ T cells (FIG. 14 b ,upper versus lower panels) and little or no IL-2Rα (FIG. 13E, left upperpanel). Strikingly, however, after activation of CD8⁺ T cells withanti-CD3+ anti-CD28, H9-RET could induce weak/partial phosphorylation ofSTAT5 (FIG. 13E, lower right panel;

FIG. 13F, lower panel; FIG. 13G), and of S6 ribosomal protein (FIG.13H), a member of the PI 3-kinase signaling pathway)), whereas H9-RETRremained essentially inert. Interestingly, H9-T significantly inducedpSTAT5 in freshly isolated CD8⁺ T cells (FIG. 13E, upper right panel),and this was markedly increased in pre-activated T cells, albeit stillnot up to levels observed with IL-2, H9, or H9-RE (FIG. 13E, lower rightpanel and FIG. 13G). Thus, H9-T and H9-RET exhibited intermediateactivities reminiscent of true partial agonists.

Given the weak pSTAT5 expression induced by H9-RET and H9-RETR, theeffects of these molecules on lymphocyte proliferation were furtherinvestigated. Whereas IL-2 and H9 strongly induced proliferation ofprimary CD8⁺ T cells and H9-T was intermediate in its effects, neitherH9-RET nor H9-RETR induced proliferation of these cells as assessed bycarboxyfluorescein diacetate succinimidyl diester (CFSE) dilution (FIG.15 ) or by [³H]-thymidine incorporation (FIG. 16A, upper panel).However, when pre-activated cells were used, H9-RETR still had noeffect, but H9-RET reproducibly induced proliferation (FIG. 16B, lowerpanel), revealing that H9-RET can induce different functional outcomesin distinct cell subsets, consistent with the differential effects ofH9-RET on pSTAT5 in freshly isolated versus pre-activated CD8⁺ T cells(FIG. 13E). As expected, IL-2 and H9 could potently induce IL-2Rαexpression in pre-activated CD8⁺ T cells, and H9-T was intermediate inits level of IL-2Rα induction, whereas H9-RET and H9-RETR significantlydecreased IL-2Rα expression, actually to below the control level,presumably reflecting their potent competition with endogenous IL-2(FIG. 17 ). RNA-Seq was next used to further elucidate the basis for theweak actions of H9-RET and H9-RETR in pre-activated CD8⁺ T cells (FIGS.16 , B and C). As expected, IL-2 and H9 induced genes that control cellcycle or are involved in cytokine signaling (e.g. CCND2, IL2RA, CISH,and CDK6) but repressed many other genes (e.g., IL7R, BCL6), whereasH9-RET had only weak stimulatory activity and H9-RETR had almost noeffect (FIG. 16B and table Si, revealing both quantitative andqualitative differences in gene expression between full and partialagonist signals. IL-2 and H9 induced more genes than they repressed,whereas H9-RETR repressed more genes than it induced, although itsoverall effect was minimal (FIG. 16C). Because STAT5 is a key mediatorof IL-2-induced transcription, chromatin immunoprecipitation and nextgeneration sequencing (ChIP-Seq) were used to globally evaluate genomicSTAT5 binding. H9-RET and H9-RETR-induced STAT5 binding to consensusTTCnnnGAA motifs, but at far fewer sites than was observed with eitherIL-2 or H9 (FIG. 16D). Based on heat map clustering, only ˜35% ofIL-2-induced STAT5 sites were also induced by H9-RET, whereas H9-RETRhad little effect (FIG. 16E). The induction (IL2RA, CISH, LTA) orrepression (IL7RA, BCL6) of several STAT5 target genes in pre-activatedCD8⁺ T cells by IL-2 and H9 was confirmed by RT-PCR (FIG. 16F), whereasneither H9-RET nor H9-RETR had an effect, except that interestingly,unlike RETR, RET reproducibly lowered BCL6 expression (FIG. 16F).

The above studies established the attenuated activity of H9-RET andnegligible activity of H9-RETR, which effectively is a dominant-negativeantagonist of IL-2. Given their enhanced binding to IL-2Rβ and abilityto decrease IL-2Rα expression on pre-activated T cells, it washypothesized that these molecules would inhibit not only endogenousIL-2, but also IL-15, which also signals via IL-2Rβ and γ_(c). Indeed,both H9-RET and H9-RETR inhibited IL-2-induced (FIG. 18A) andIL-15-induced (FIG. 18B) pSTAT5 in CD8⁺ T cells, with H9-RETR being morepotent. Inhibition of pSTAT5 by H9-RET and H9-RETR treatment correlatedwith their lowering TCR-induced (FIG. 17 ) and IL-2-induced (FIG. 19A)CD25 expression to levels below those observed in unstimulated controlcells. Correspondingly, H9-RET and H9-RETR inhibited IL-2-induced IL2RAmRNA expression (FIG. 19B) as well as IL-2- and IL-15-inducedproliferation (FIG. 19C) in pre-activated human CD8⁺ T cells.

Because H9-RETR inhibited IL-2 signaling, it speculated that H9-RETRwould also inhibit TCR-induced cell proliferation, which is dependent onIL-2, and this was indeed the case (FIG. 20A) and correlated withdiminished CD25 expression (FIG. 20B). Similarly, as IL-2 can promoteTh1, Th9, and Treg differentiation but inhibit Th17 differentiation(Liao et al., Immunity 38: 13 (2013)), the effects of H9-RET and H9-RETRon these processes were examined. Strikingly, both H9-RET and H9-RETRinhibited Th1, Th9, and Treg differentiation but augmented Th17differentiation (FIG. 21 ), underscoring their actions as potentantagonists of IL-2.

The activation of primary human NK cells by IL-2 was also potentlyblocked by H9-RETR, as measured by IL-2-induced CD69 expression (FIG.22A) and cytotoxicity towards the breast cancer cell line HER18 (FIG.22B) and the chronic myelogenous leukemia cell line K562 (FIG. 22C).Neither H9-RET nor H9-RETR stimulated CD69 expression or cytotoxicity byprimary NK cells (FIG. 22A).

Example 12: Partial IL-2 Agonists and Antagonists—In Vivo Effects

Given H9-RETR's effectiveness as an IL-2/IL-15 antagonist in vitro, weinvestigated its ability to antagonize the effects of endogenouscytokines in vivo. Because IL-2 has a short serum half-life (Boyman etal., Nature Reviews Immunology 12: 180 (2012)), H9-RETR was fused to theFc fragment of human IgG4 (Fc4), an isotype with diminishedantibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP)(Strohl, Current Opinion in Biotechnology 20: 685 (2009)). Strikingly,as compared to anti-Tac mAb to CD25 and Mikβ1 mAb to IL-2Rβ (Morris etal., Proc Natl Acad Sci USA 103: 401 (2006)), H9-RETR-Fc4 much morepotently blocked IL-2- (FIG. 18C) and IL-15-mediated (FIG. 18D) pSTAT5induction in pre-activated human CD8⁺ T cells and inhibited IL-2- (FIG.18E) and IL-15- (FIG. 18F) induced proliferation of human CD8⁺ T cells.Importantly, H9-RETR-Fc4 was as effective as the combination of anti-Tacand Mikβ1 in blocking IL-2 proliferation (FIG. 18E) and more potent thanMikβ1 in inhibiting IL-15-induced proliferation (FIG. 18F).

The effectiveness of H9-RETR-Fc in vivo was next evaluated. Understeady-state conditions, Treg cells are the dominant population ofprimary cells expressing high affinity IL-2 receptors and can act as asystemic ‘barometer’ of IL-2 signaling. Pre-treatment of mice withH9-RETR-Fc4 prior to administering IL-2 or IL-15 significantly inhibitedphosphorylation of STAT5 in CD4⁺FoxP3⁺ Treg cells, as assessed ex vivo(FIG. 23 and FIG. 18G), indicating the in vivo potential of H9-RETR-Fc4as an IL-2 antagonist. Because IL-2 and IL-15 signaling contributes toacute GVHD in experimental murine models (Ferrara et al., Journal ofImmunology 137: 1874 (1986); and Blaser et al., Blood 105: 894 (2005)),it was hypothesized that H9-RETR-Fc4 might inhibit lethal GVHD in a Tcell-mediated C57BL/6-into-BALB/C model of fully-MHC mismatched bonemarrow transplantation. Indeed, mice treated for 10 days withH9-RETR-Fc4 had longer survival than mice treated with isotype controlFc4 protein (P<0.001) (FIG. 18H).

Human T-cell lymphotropic virus-I (HTLV-I) causes adult T-cell leukemia(ATL), a malignant expansion of CD4⁺ T cells that exhibit an earlygrowth phase that involves autocrine signals by IL-2 and IL-15 andparacrine signals by IL-9. Such cytokine-dependent proliferation isevident in patients with chronic and smoldering but not acute ATL (Ju etal., Blood 117, 1938 (2011)). As such the efficacy of H9-RETR in thissystem was tested. An ATL-derived cell line, ED40515, whose growth issupported in vitro by adding exogenous IL-2, was first used. H9-RETRpotently inhibited proliferation of these cells, and was superior todaclizumab and Mikβ1 (FIG. 18I). There, cells freshly isolated from apatient with smoldering ATL were next used and assayed for spontaneousproliferation in a six day assay. RETR at 10 μg/ml was slightly moreeffective than daclizumab and considerably more effective than Mikβ1(FIG. 18J), underscoring its potential utility in the control of thesevigorously proliferating malignant cells.

Partial agonism, defined as reduced signaling amplitude (E_(max)) atligand saturation, is a pharmacological property typically associatedwith small molecules targeting GPCRs, channels, and other multi-passtransmembrane proteins. The notion of tunable signaling (partialagonism) through a type I transmembrane receptor dimer in response to aprotein growth factor such as a cytokine has not been demonstrated.Based on structural information, IL-2 variants were engineered byenhancing affinity at one receptor binding site (IL-2Rβ), whileattenuating interactions at the second receptor binding site (γ_(c)) inorder to manipulate dimerization and signal initiation. Because ofaugmented IL-2Rβ binding, these molecules were dominant over endogenousIL-2, and their levels of γ_(c) interaction set the intensity of thesignal appreciated by the cell, thereby “clamping” signaling amplitudeat the level of the partial agonist's Emax. Using these partialagonists, it was demonstrated that freshly-isolated versus preactivatedCD8⁺ T cells had distinct activation thresholds for IL-2 signalingstrength as evidenced by differential effects of H9-T and H9-RET onthese cells, whereas H9-RETR, which is an extremely weak partial agonistas shown by pSTAT induction, had marked inhibitory properties, withpotential as a novel type of immunosuppressive agent. In particular, itcould block IL-2Rα induction as assessed ex vivo, prolonged survival ina GVHD model, and potently inhibited the spontaneous proliferation ofperipheral blood T cells from a patient with smoldering ATL. Besides itseffects on T cells, H9-RETR also inhibited NK-mediated cytotoxicity.Given the differential activities of the partial agonists, a largerrepertoire of IL-2 variants may reveal an even broader spectrum ofdistinctive signaling activities, ranging from partial agonism tocomplete antagonism, and potentially including additional molecules withdistinctive actions on T cell subsets, such as Treg versus effector Tcells. Moreover, the rational design approach we have used can beadapted to other γ_(c) family cytokines, and indeed for a broader rangeof cytokines and growth factors as well. Additional partial agonistcytokine analogues potentially could dissect the functions of otherwisepleiotropic immune pathways and have distinctive therapeutic benefitsdepending on the context.

Material and Methods

Protein Expression and Purification

Human IL-2 (amino acids 1-133) and variants thereof, the human IL-2Rβectodomain (amino acids 1-214), and the γ_(c) ectodomain (amino acids34-232), were secreted and purified using a baculovirus expressionsystem, as previously described (Morgan et al., Science 193: 1007 (Sep.10, 1976)). In brief, all construct sequences were cloned into thepAcGP67A vector (BD Biosciences) with an N-terminal gp67 signal peptideand a C-terminal hexahistidine tag. Spodoptera frugiperda (Sf9) insectcells cultured at 28° C. in SF900 II SFM medium (Invitrogen) weretransfected with the plasmid constructs to establish high titerrecombinant virus, which was subsequently amplified. Trichopulsia ni(High-Five®) insect cells (Invitrogen) grown in Insect Xpress medium(Lonza) at 28° C. were infected with the high titer viruses to expressrecombinant protein (Zhu et al., Annual Review of Immunology 28: 445(2010) and W. Liao et al., Nat Immunol 9: 1288 (2008)). Three days afterinfection with recombinant virus, proteins were extracted via Ni-NTA(Qiagen) affinity chromatography, concentrated, and further purifiedto >98% homogeneity with a Superdex 200 sizing column (GE Healthcare)equilibrated in 10 mM HEPES (pH 7.3) and 150 mM NaCl. Fc4 and Fc4-RETRfusion proteins were also secreted and purified using this baculovirusexpression system by cloning the human IgG4 Fc domain (Fc4) or the humanIL-2 RETR variant followed by a C-terminal human IgG4 Fc domain(Fc4-RETR) into the pAcGP67A vector containing an N-terminal gp67 signalpeptide and a C-terminal hexahistidine tag. The human IgG4 Fc domain wasobtained from a modified pFUSE-hIgG4-Fc vector (Invivogen) with anengineered Ser228 Pro mutation (Liao et al., Nat Immunol 12: 551(2011)). For in vivo experiments, endotoxin was removed from preparedproteins using Triton X-114 as previously described (Cheng at al.,Immunol Rev 241: 63 (2011) and endotoxin removal was verified with theLAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific).

For biotinylated protein expression, γ_(c) with a C-terminal biotinacceptor peptide (BAP)-LNDIFEAQKIEWHE was expressed and purified viaNi-NTA (Qiagen) affinity chromatography and then biotinylated with thesoluble BirA ligase enzyme in 0.5 mM Bicine pH 8.3, 100 mM ATP, 100 mMmagnesium acetate, and 500 mM biotin (Sigma). Proteins were purified bysize exclusion chromatography on a Superdex 200 column (GE Healthcare),equilibrated in 10 mM HEPES (pH 7.3) and 150 mM NaCl.

Surface Plasmon Resonance Binding Measurements

Binding interactions were characterized via surface plasmon resonance(SPR) studies using Biacore SA sensor chips (GE Healthcare) on a BiacoreT100 instrument. γ_(c) was immobilized to the chip surface at lowdensity (RU_(max)<100 response units), and serial dilutions of H9:IL-2Rβor H9-RET:IL-2Rβ complexes were exposed to the surface for 60 s.Dissociation was then tracked for 200 s. An irrelevant biotinylatedprotein was immobilized in the reference channel to subtractnon-specific binding from the measurements. Experiments were carried outin HBS-P+ buffer (GE Healthcare) supplemented with 0.2% BSA at 25° C.All binding studies were performed at a flow rate of 30 mL/min tominimize mass transport contributions and prevent rebinding of theanalyte. For all measurements, data analysis and determination ofequilibrium and kinetic parameters were implemented using the BiacoreT100 evaluation software version 2.0 assuming a 1:1 Langmuir bindingmodel.

Tissue Culture and Magnetic Purification of CD25⁺ YT-1 Cells

Unmodified YT 9 (Zhu et al., Annual Review of Immunology 28: 445 (2010))and CD25⁺ YT-1 natural killer-like cells (Liao et al., Nat Immunol 9:1288 (2008)) were cultured in RPMI complete medium (RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 2 mM L-glutamine, minimumnon-essential amino acids, sodium pyruvate, 25 mM HEPES, andpenicillin-streptomycin (Gibco)). Both cell lines were maintained at 37°C. in a humidified atmosphere with 5% CO2.

Subpopulations of YT-1 cells expressing or not expressing CD25 werepurified via magnetic selection, as detailed previously (Liao et al.,Immunity 38: 13 (2013)). Ten million unsorted CD25⁺ YT-1 cells werewashed with FACS buffer (phosphate-buffered saline pH 7.2 containing0.1% bovine serum albumin) and subsequently incubated with PE-conjugatedanti-human CD25 antibody (Biolegend, clone BC96) in FACS buffer for 2 hrat 4° C. The PE-stained CD25⁺ cells were then labeled with paramagneticmicrobeads conjugated to an anti-PE IgG for 20 min at 4° C., washed oncewith cold FACS buffer, and sorted using an LS MACS separation column(Miltenyi Biotec) according to the manufacturer's protocol. Eluted cellswere re-suspended and grown in RPMI complete medium and enrichment ofCD25⁺ cells was evaluated using an Accuri C6 flow cytometer. Persistenceof CD25 expression on the sorted CD25⁺ YT-1 cells was monitored by flowcytometric analysis using PE-conjugated anti-human CD25 antibody.

Flow cytometric analysis of intracellular phospho-STAT5 andphospho-ERK1/2 Approximately 2×10⁵ YT or CD25⁺ YT-1 cells were plated ineach well of a 96-well plate, washed with FACS buffer, and re-suspendedin FACS buffer containing serial dilutions of IL-2, H9, H9-RET, orH9-RETR. Cells were stimulated for 20 min at 37° C. and immediatelyfixed by addition of formaldehyde to 1.5% followed by incubation for 10min at room temperature. Cells were then permeabilized with 100%ice-cold methanol for 30 min at 4° C. to allow for detection ofintracellular signal effectors. The fixed and permeabilized cells werewashed twice with FACS buffer and incubated with Alexa488-conjugatedanti-STAT5 pY694 (BD Biosciences) or Alexa488-conjugated anti-ERK1/2pT202/pY204 (BD Biosciences) diluted in FACS buffer for 2 hr at roomtemperature. Cells were then washed twice in FACS buffer and meanfluorescence intensity (MFI) was quantified on an Accuri C6 flowcytometer (BD Biosciences). Dose-response curves were generated, andEC₅₀ and E_(max) values were calculated using the GraphPad Prism dataanalysis software after subtraction of the MFI of unstimulated cells andnormalization to the maximum signal intensity induced by cytokinestimulation.

Human CD8⁺ T Cell Isolation and Intracellular Staining of pSTAT5 andpS6-Ribosomal Protein

Buffy coats were obtained from healthy donors from the NIH Blood Bank,and peripheral blood mononuclear cells (PBMCs) were isolated by gradientcentrifugation using lymphocyte separation medium (Mediatech, Inc., VA).Cells were isolated using the human CD8⁺ T-cell Isolation kit I(Miltenyi Biotec, Germany). For pre-activating cells, 6-well plates werepre-coated with 2 μg/ml of plate-bound anti-CD3 mAb (BD Biosciences).Cells were seeded at 1×10⁶ cells/ml in complete medium (RPMI mediumsupplemented with glutamine, penicillin, streptomycin, and 10% FBS) with1 μg/ml soluble anti-CD28 mAb (BD Biosciences) for 3 days and thenrested for 48 h in fresh medium. Dose-response experiments on primaryhuman CD8⁺ T cells were performed as previously described (Liao et al.,Immunity 38: 13 (2013)); in brief, cells were treated with serialdilutions of IL-2, H9, H9-RET, H9-RETR, then fixed with Phosflow FixBuffer I at room temperature for 10 minutes (BD Biosciences), and washedonce with FACS buffer. Cells were then permeabilized by slowly addingcold BD Phosflow™ Perm Buffer III and incubated for 30 min on ice. Cellswere washed and stained with PE-conjugated anti-STAT5 pY694 (BDBiosciences) or APC-conjugated anti-phospho-S6 ribosomal protein(Ser235/236) (clone D57.2.2E) at room temperature for 30 min in thedark, washed again with FACS buffer, and data acquired on a FACSCanto IIflow cytometer (BD Biosciences) and analyzed by using FlowJo (TreeStar).

Analysis of STAT5 Phosphorylation Ex Vivo

C57BL/6 mice were from the Jackson Laboratory. Animal protocols wereapproved by the NHLBI Animal Care and Use Committee and followed the NIHGuidelines, “Using Animals in Intramural Research.” STAT5phosphorylation was assayed using the manufacturer's protocol (BDBioscience). In brief, IL-2 or IL-15 were injected i.p. into C57BL/6mice, total splenocytes isolated, immediately fixed using BDPhosphoflow™ Lyse/Fix buffer, washed twice with ice cold PBS, stainedusing anti-CD4 and anti-CD25 antibodies (Biolegend), and thenpermeabilized using BD PhosFlow Perm Buffer III for 30 min on ice in thedark. Cells were then washed twice with ice-cold FACS buffer, stainedwith anti-FoxP3 per the manufacturer's protocol (eBioscience), washedtwice with ice-cold FACS buffer, and stained with anti-phospho-STAT5-PE(1:30) (BD) at room temperature for 30 min in the dark. Cells werewashed three times with FACS buffer, and data acquired on a FACS Cantoflow cytometer (BD) and analyzed using FlowJo (Tree Star).

IL-2 Receptor Internalization Experiments

IL-2, H9, H9-RET, or H9 RETR (1 μM) was incubated with 3×10⁵ YT-1 cellsin a 96-well plate for 2, 5, 10, 15, 30, 60, 90, 120, 180, or 240 min.Cells were immediately transferred to ice to prevent further receptorinternalization and washed twice with ice-cold PBSA buffer (0.1% BSA inPBS). Cells were concurrently stained with 1:50 dilutions ofallophycocyanin-conjugated anti-human IL-2Rβ antibody (TU27; Biolegend)and phycoerythrin-conjugated anti-human IL-2Rγ antibody (TUGh4;Biolegend) in PBSA buffer for 30 min on ice. After two more washes inice-cold PBSA buffer, cells were fixed for 10 min at room temperaturewith 1.5% paraformaldehyde in PBSA, washed one final time, andresuspended in PBSA buffer. Mean cell fluorescence was quantified withan Accuri C6 flow cytometer. Internalization data were fitted to asingle exponential decay model using non-linear least squares regressionwith the Prism software package (GraphPad).

Inhibition of IL-2-Induced pSTAT5

Freshly isolated and pre-activated human CD8⁺ T-cells were stimulatedwith IL-2 in the absence or presence of H9-RET or H9-RETR, and pSTAT5levels assessed. Cells were incubated with or without anti-Tac or Mikβ1,or Fc4-H9-RETR for 1 hr, then stimulated with a range of doses of IL-2or IL-15 for 30 min, and pSTAT5 levels measured by flow cytometry. ForNK cell experiments, freshly isolated human NK cells (NK Cell IsolationKit II, Miltenyi Biotech) were stimulated with serial dilutions of IL-15in the presence or absence of the indicated IL-2 variant, and pSTAT5assessed.

Western Blot Analysis

Cells stimulated with or without cytokines were lysed in RIPA buffercontaining 1% IGEPAL CA-630 (Sigma). Equal amounts of lysates wereresolved on 4 to 12% Bis-Tris NuPAGE gels (Invitrogen), transferred tomembranes, and the membranes incubated for 1 h at room temperature withantibodies to pSTAT5(Y694) (Cell Signaling Technology, Inc., Beverly,Mass.) or STAT5 (BD Transduction Laboratories, San Diego, Calif.). Boundantibodies were detected with goat anti-rabbit-IgG (H+L)-HRP conjugate(1:5,000 dilution) and with goat anti-mouse IgG (H+L)-HRP conjugate(1:10,000 dilution) (Biorad). Immunoblots were visualized using enhancedchemiluminescence (ECL, GE healthcare). In some experiments, membraneswere reused after incubating in stripping buffer (Millipore) for 15 minat room temperature.

CFSE Dilution and EdU Proliferation Assays

Freshly isolated or pre-activated CD8⁺ T cells (20×10⁶/ml) were labeledwith 2.5 μM CFSE (Molecular Probes) in PBS at room temperature for 7 minand immediately washed once with 100% FBS (2 ml/sample) and then twicein complete RMPI. 2×10⁶/ml CFSE labeled cells were cultured in theabsence or presence of wild type IL-2, H9, H9-RET, H9-RETR, or IL-2 plusH9-RETR. Cell proliferation was assessed by flow cytometric analysis ofCFSE dilution at indicated time-points. For EdU proliferation assays,pre-activated CD8⁺ T cells were cultured as described above. 16 h beforeharvesting, 10 mM EdU was added, cells were stained for surface antigensas indicated, and then for intracellular EdU according to themanufacturer's protocol (BD Biosciences). Cell proliferation wasassessed by flow cytometry.

Proliferation of ED40515 Cells and Cells from a Patient with SmolderingATL

IL-2-dependent ED40515(+) (Lenardo, Nature 353: 858 (1991)) cells werewashed twice with PBS. Cells were seeded into 96-well plates, at 1×10⁴cells/100 μl/well of RPMI 1640 medium with or without IL-2 and with orwithout reagents, and then incubated at 37° C. for 3 days.

Blood samples from ATL patient were obtained under the care of theClinical Trials Team, Lymphoid Malignancies Branch, NCI, NIH. This studyprotocol was approved by the Institutional Review Board of the NationalCancer Institute. Informed consent was obtained in accordance with theDeclaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) fromATL patients were separated by Ficoll-Hypaque density gradientcentrifugation from their heparinized blood. Aliquots of 1×10⁵ cells/100μl/well were cultured ex vivo in RPMI 1640 medium supplemented with 10%FBS with or without reagents for 6 days.

During the last 6 hours of culture, ED40515(+) cells or ATL PBMCs werepulsed with 1 μCi (0.037 MBq) [³H]thymidine, then the cells wereharvested with a cell harvester (Tomtec, Hamden, Conn.) and counted witha MicroBeta2 microplate counter (PerkEmer). The assay was performed intriplicate.

Activation of NK cells

Peripheral blood mononuclear cells (PBMCs) were cultured in the presenceof 1 μg/mL IL-2 analogues for 24 hours. Cells were then stained withAPC-conjugated anti-CD56 (BD Biosciences), Pacific Blue-conjugatedanti-CD3 (BioLegend), and FITC-conjugated anti-CD69 (BD Biosciences).Samples were analyzed by flow cytometry using a FACSAria II (BDBiosciences). NK cells were gated as CD3-negative, CD56-positive.

PBMCs were isolated by gradient centrifugation using Ficoll-PaquePremium (GE Life Sciences), then untouched NK cells were purified usingthe Human NK Cell Isolation Kit (Miltenyi) followed by separation usingan autoMACS (Miltenyi). HER18 target cells were labeled with 150 μCi⁵¹Cr (Perkin Elmer) per 1×10⁶ cells for 2 hours. NK cells were added to10,000 HER18 cells at a 10:1 effector:target ratio. Specific lysis wasdetermined after 4 hours of culture in the presence of varyingconcentrations of IL-2 analogues.

Lysis of K562 cells by primary NK cells was performed as described (Yuanet al., Immunol Rev 259, 103 (2014)). In brief, untouched human NK cellswere purified using the human NK isolation kit (STEMCELL). K562 cellswere labeled with the PKH67 green fluorescent cell linker kit(SigmaAldrich), NK cells were added to 5,000 K562 cells at a 10:1 ratio,cultured in the presence of varying concentrations of IL-2 analogues at37° C. for 4 h, and placed on ice to prevent further reactivity. Cellswere then stained with propidium iodide (PI)(Sigma-Aldrich), and thepercentage of PI⁺ PKH67⁺ K562 cells was determined by flow cytometry.

T-Helper Polarization and Intracellular Cytokine Staining

Naïve CD4⁺ C57BL6 T-cells were differentiated under different T-helperpolarization conditions in the absence or presence of the indicatedcytokines. Four days later, cells were first stained for surfaceantigens as indicated and then stained with antibodies to IFNγ(eBioscience), IL-17A (eBioscience), IL-4 (BioLegend), IL-9 (BioLegend),or FoxP3 (eBioscience) in BD cytofix and cytoperm, or eBioscience FoxP3staining buffer kit according to the manufacturer's protocol. Stainedcells were analyzed on a FACSCanto II flow cytometer (Becton Dickinson)using FlowJo software (Tree Star, Inc). All mouse cytokines were fromPeprotech.

RNA-Seq Analysis

Pre-activated human CD8⁺ T-cells were rested for two days in completemedium, stimulated for 24 hr with 1 μg/ml of wild type IL-2, H9, H9-RET,or H9-RETR, and total RNA from 5×10⁶ cells was isolated (RNeasy kit,Qiagen, Valencia, Calif.). RNA from 5 donors was pooled, and 1 μg of thepooled RNA was used to synthesize cDNA. RNA-seq libraries were preparedas described previously (Liao et al., Immunity 38: 13 (2013). PCRamplified products were barcoded and sequenced using an IlluminaHiSeq2000 platform. Sequenced reads were aligned against the humangenome (hg18, NCBI build 36.1) using Bowtie 0.12.4 (Leonard, NatureReviews. Immunology 1: 200 (2001)). Uniquely mapped reads were retained,and digital expression levels of genes were calculated using RPKM (ReadsPer Kilobase per Million mapped reads). R package edgeR was used toidentify differentially expressed genes, and fold-change (in log 2scale) differences were compared between cells not treated or treatedwith the IL-2 variants for 24 h.

ChIP-Seq Library Preparation and Analysis

Pre-activated CD8⁺ T-cells were treated with different cytokines for 90min and then chemically cross-linked with 1% paraformaldehyde. Chromatinfrom 10-20 million cells was sonicated into 250-500 bp fragments,immunoprecipitated with anti-STAT5B (Invitrogen) and processed forsequencing as described previously (Noguchi et al., Cell 73: 147(1993)). All reads were aligned against the human genome (hg18, NCBIbuild 36.1) using Bowtie 0.12.4 (Leonard, Nature Reviews. Immunology1:200 (2001). Uniquely mapped reads were converted to browser extensibledata (BED) files and duplicated reads (multiple reads in same genomiclocation) were filtered. The filtered (non-redundant) BED files werethen converted to binary tiled data (.tdf) and visualized using theIntegrative Genome Viewer (Broad Institute).

Gene Expression Analysis by RT-PCR

Total RNA was isolated using RNeasy Plus mini kit (Qiagen) and 200 ngwas used together with oligo dT (Invitrogen) and the Omniscript reversetranscription kit (Quiagen) to synthesize cDNA. Quantitative RT-PCR wasperformed with an ABI 7900 HD Sequence Detection System. RT primers andprobes were from Applied Biosystems. Expression levels were normalizedto RPL7.

Bone Marrow Transplantation into Allogeneic Hosts

7 week old female C57BL/6 (H₂K^(b)) and BALB/c (H₂K^(d)) mice from theNCI-Frederick Cancer Research Facility were maintained in a specificpathogen-free facility and treated according to an approved animalprotocol approved by the NCI Animal Care and Use Committee. BALB/c micewere conditioned with 950 cGy total body irradiation and thenreconstituted with 10 million T-cell depleted bone marrow cells fromC57BL/6 mice alone or together with 2 million Treg-depleted pan-T cells.T cell depletion was performed with anti-CD90 [Thy1.2] microbeads (totalT cell depletion) or anti-CD25 (Treg depletion) using kits from MiltenyiBiotec. Mice receiving pan-T cells were additionally treated with Fc4 orH9-RETR-Fc4 (100 μg i.p. twice/day for 10 days). Drinking water wassupplemented with ciprofloxacin from day −1 to +14 after total bodyirradiation. Survival and weight loss were monitored. Survival wasanalyzed according to the Kaplan-Meier method, and survival curves werecompared using the log-rank test. Statistical analysis was performedusing GraphPad Prism 4 software.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. For example, although IL-2 is referred to throughoutthe specification, one of skill in the art would appreciate that themethods and compositions described herein are equally applicable toother cytokines, for example, granulocyte-macrophage colony-stimulatingfactor (GM-CSF), IL-2, IL-3, IL-5, IL-6, or IL-15 with this property.Thus, the invention also includes mutants of GM-CSF, IL-2, IL-3, IL-5,IL-6, and IL-15 with increased binding affinity for their respectivereceptors, as compared to wild-type, and methods for identifying andusing those mutants.

1-36. (canceled)
 37. A nucleic acid encoding an IL-2 mutein having anincreased binding affinity for IL-2Rβ and a decreased binding affinityfor IL-2Rγc receptor as compared to wild-type human IL-2 (hIL-2),wherein said IL-2 mutein comprises amino acid substitutions L18R, Q22E,Q126T, and S130R, numbered in accordance with wild-type hIL-2.
 38. Thenucleic acid of claim 37, wherein the IL-2 mutein further comprises oneor more amino acid substitutions that increase IL-2Rβ binding affinity,wherein the one or more amino acid substitutions that increase IL-2Rβbinding affinity are selected from the group consisting of: Q74N, Q74H,Q74S, L80F, L8V, R81D, R81T, L85V, 186V, 189V, and 193V.
 39. The IL-2mutein of claim 2, wherein the IL-2 mutein comprises amino acidsubstitutions L80F, R81D, L85V, 186V, and 192F.
 40. The IL-2 mutein ofclaim 2, wherein the IL-2 mutein comprises amino acid substitutionsQ74N, L80F, R81D, L85V, 186V, 189V, and 192F.
 41. The IL-2 mutein ofclaim 2, wherein the IL-2 mutein comprises amino acid substitutionsQ74N, L80V, R81T, L85V, 186V, and 192F.
 42. The IL-2 mutein of claim 2,wherein the IL-2 mutein comprises amino acid substitutions Q74H, L80F,R81D, L85V, 186V, and 192F.
 43. The IL-2 mutein of claim 2, wherein theIL-2 mutein comprises amino acid substitutions Q74S, L80F, R81D, L85V,186V, and 192F.
 44. The IL-2 mutein of claim 2, wherein the IL-2 muteincomprises amino acid substitutions Q74N, L80F, R81D, L85V, 186V, and192F.
 45. The IL-2 mutein of claim 2, wherein the IL-2 mutein comprisesamino acid substitutions Q74S, R81T, L85V, and 192F.
 46. The nucleicacid of claim 37, wherein the mutein has a decreased capability tostimulate STAT5 phosphorylation in an IL-2Rβ+ T cell as compared towild-type hIL-2.
 47. The nucleic acid of claim 46, wherein the T cell isa CD8+ T cell.
 48. The nucleic acid of claim 37, wherein the mutein hasa decreased capability to stimulate the pERK1/ERK2 signaling in aIL-2Rβ+ cell as compared to wild-type hIL-2.
 49. The nucleic acid ofclaim 37, wherein the mutein is an IL-2 or IL-15 antagonist.
 50. Thenucleic acid of claim 49, wherein the mutein is an inhibitor of IL-2 orIL-15 STAT5 phosphorylation in CD8+ T cells.
 51. The nucleic acid ofclaim 49, wherein the mutein is an inhibitor of IL-2 or IL-15 inducedproliferation of CD8+ T cells.
 52. The nucleic acid of claim 49, whereinthe mutein is an inhibitor of IL-2 dependent, TCR-induced cellproliferation.
 53. The nucleic acid of claim 49, wherein the mutein isan inhibitor of IL-2 dependent Th1, Th9 or Treg differentiation.
 54. Thenucleic acid of claim 49, wherein the mutein is a promoter of Th17differentiation.
 55. The nucleic acid of claim 49, wherein the mutein isan inhibitor of IL-2 dependent activation of NK cells.
 56. A nucleicacid encoding an IL-2 mutein fusion protein comprising the IL-2 muteinof claim 37 and a human Fc antibody fragment.
 57. A nucleic acidencoding an IL-2 mutein fusion protein comprising the IL-2 mutein ofclaim 37 and a heterologous polypeptide.
 58. A nucleic acid encoding anIL-2 mutein fusion protein comprising the IL-2 mutein of claim 37 and analbumin polypeptide.